Thyroid hormones play an essential role in metabolism, growth and development. Thyroid hormone metabolism was considered to be important for recycling of iodine and clearance of thyroid hormone metabolites, but the thyroid hormone metabolites itself were regarded to be inactive. Nowadays, thyroid hormone metabolites downstream from the two thyroid hormones T3 and T4 are being suggested to have several physiological roles. To gain insight into the clinical effects of the metabolites and to determine whether thyroid hormone metabolites give us meaningful information to better understand pathophysiological processes, we developed a multi-analyte assay using mass spectrometry. The metabolites that were combined in the multi-analyte assay was determined by the clinical question we wanted to answer and the structural and chemical properties of the metabolites. The research which is presented in this thesis focuses on the method development, including analytical and clinical validation, to quantify total concentrations of two thyroid hormones and seven thyroid hormone metabolites in a single run using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated according to CLSI and EMA guidelines. The clinical validation was performed in a cohort of healthy individuals, patients with differentiated thyroid cancer and patients with acute coronary syndrome. Chapter 1 provides a general introduction on thyroid hormone metabolism, the physiological roles attributed to the studied thyroid hormone metabolites and the different analytical techniques used to measure thyroid hormones in healthy individuals. Chapter 2 focuses on sample preparation strategies for quantification of thyroid hormones and thyroid hormone metabolites by targeted LC-MS/MS and describe a new mass spectrometric method for the quantification of seven iodothyronines and two iodothyroacetic acids in human serum. Chapter 2 Part A highlights sample preparation strategies, since method development of a multi-analyte assay is challenging due to diverse chemical properties of the metabolites and broad concentration ranges (picomolar to millimolar) among metabolites. We discuss protein precipitation, liquid-liquid extraction and solid phase extraction and also focus on other key factors to consider, such as adsorptive loss to standard laboratory ware, availability of analyte-free biological matrices, internal standards, reconstitution and stability during sample preparation. We advise to take four considerations into account, when developing a multi-analyte assay for thyroid hormone metabolites. First, use glass vials and glass inserts for single-use due to adsorptive loss to polypropylene and to minimize cross-contamination. Second, use an extensive and selective sample preparation strategy e.g. combine a protein precipitation step with a solid phase extraction step with a mixed-mode sorbent to increase selectivity and obtain a clean sample. Third, check the internal standard for impurities of the non-labeled compound and of other non-labeled and labeled compounds that are quantified in the metabolite panel. Fourth, determine the needed equilibration time of the internal standard to ensure proper correction of losses during the entire sample preparation. In Chapter 2 Part B, the method development is described of a mass spectrometry-based assay to quantify seven iodothyronines and two iodothyroacetic acids in human serum in a single run. Subsequently, reference values were determined in healthy individuals. Extensive sample preparation and the use of 13C6-labelled internal standards for eight out of nine thyroid hormones and its metabolites was very important for a sensitive and robust analytical method. This LC-MS/MS method is unique as it contains three additional thyroid hormone metabolites compared to previously published thyroid hormone metabolite panels and combines two metabolic pathways, iodothyronines and iodothyroacetic acids. We found lower concentrations for 3,5-T2 and 3,3′-T2 in healthy individuals with our method compared to previously reported concentrations measured with LC-MS/MS. In our reference group, TA3 could not be detected and a large variation in TA4 concentrations was found. A large TA4 variation has also been reported in other euthyroid patient cohorts. In Chapter 3, we focused on the binding characteristics of thyroid hormone metabolites with thyroid hormone distributor proteins. In addition, we determined the association of thyroid hormone distributor proteins with thyroid hormones and thyroid hormone metabolites using in vivo concentrations. We found that the distribution of thyroid hormone metabolites between distributor proteins differs from that of T4 and T3, which predominantly bind to TBG. The predominant distributor protein of 3,3′- T2 and rT3 is albumin, of TA3 is TTR and albumin and of TA4 is TTR. For TBG, the rank order of affinity was T4>TA4=rT3>T3>TA3=3,3′-T2>3-T1=3,5-T2>T0 (IC50- range: 0.36 nM to > 100 μM) and for TTR the rank order of affinity was TA4>T4=TA3>rT3>T3>3,3′-T2>3-T1>3,5-T2>T0 (IC50-range: 0.94 nM to >100 μM). A positive association was found for TBG with T3 and T4, but not for TTR or albumin with T3 and T4. TBG, TTR and albumin were not associated with T0, 3-T1, 3,3′-T2, rT3 and TA4. Therefore, serum TBG, TTR and albumin concentrations within the reference interval do not influence serum concentrations of T0, 3-T1, 3,3′-T2, rT3 and TA4. The clinical validation of the method is described in Chapters 4 to 6. In Chapter 4, we investigated thyroid hormone metabolite concentrations across different thyroid states in a cohort of patients treated for differentiated thyroid cancer. We used this patient cohort to answer two questions; 1) how does thyroidectomy affect thyroid hormone metabolite concentrations? and 2) does LT4 supplementation therapy restore thyroid hormone metabolite concentrations in patients without a thyroid gland? In our study, we found that thyroidectomy causes a decrease in all thyroid hormone and thyroid hormone metabolite concentrations. Additionally, we found that LT4 supplementation therapy restores thyroid hormone metabolite concentrations following the same trend as T4. The latter suggests that thyroid hormone metabolites in general are predominantly formed via peripheral extrathyroidal metabolism. In patients without a thyroid gland, T3 production is also solely dependent on peripheral extrathyroidal metabolism. Consequently, more LT4 should be supplemented until subclinical hyperthyroid levels are reached to ensure that T3 is within the reference interval. In Chapter 5, we studied the relationship (cross-sectionally and longitudinally) between thyroid hormones and its metabolites and quality of life across different thyroid states. In addition, we sought to identify a specific thyroid hormone metabolite that can explain the persistent symptoms in patients with hypothyroidism on LT4 replacement therapy. With the cross-sectional analysis, we did not find a relation between thyroid hormones and thyroid hormone metabolites with Quality of Life at any of the visits. With the longitudinal analysis, in general higher concentrations of T0, 3-T1, 3,3′-T2, T3, rT3 and TA4 were associated with significantly less complaints in specific Quality of Life domains compared to those with lower concentrations of these metabolites. However, we did not find any metabolite responsible for the persistent complaints in hypothyroid patients on LT4 replacement therapy. In Chapter 6, we determined whether thyroid hormone metabolite concentrations and thyroid hormone metabolite ratios are associated with an increased risk of major adverse cardiovascular events and mortality in patients admitted for acute coronary syndrome. The results show that higher T3/T4, T3/rT3 and T4/rT3 ratios are associated with a lower risk of major adverse cardiovascular events and mortality in the unadjusted model. When adjusting for age and sex the associations of T4/rT3 ratio with major adverse cardiovascular events and T3/T4 ratio and T4/rT3 ratio with mortality lost significance. A higher rT3/3,3′-T2 ratio is associated with a higher risk of major adverse cardiovascular events and mortality, but lost statistical significance when adjusting for age and sex. In non-thyroidal illness, D1 is downregulated and D3 is upregulated. Our results on thyroid hormone metabolite ratios indicate that the thyroid hormone metabolite pattern in patients admitted for acute coronary syndrome resembles non-thyroidal illness. In addition, higher T0 concentrations are associated with an increased risk of major adverse cardiovascular events and mortality in these patients. Finally in Chapter 7, we discuss the results of the studies presented in this thesis, together with the possible implications of these studies, the limitations of the studies as well as future perspectives.