The current diagnostic process for Lyme disease (Lyme borreliosis, LB) relies on clinical characteristics, supplemented with laboratory diagnostics when necessary. Laboratory work-up usually takes the form of serological testing for Borrelia burgdorferi sensu lato antibodies. The serodiagnosis of LB comes with several drawbacks, such as a limited sensitivity in early disease (erythema migrans) and the inability to differentiate an active from a past infection. This thesis addresses the diagnostic parameters of various test modalities for LB that seek to circumvent these drawbacks. Through retrospective studies on samples from various serum banks, my co-authors and I were able to show that serological testing for LB may be improved in various ways. We confirmed that combining validated serological tests in a novel way (modified two-tier testing) improves the sensitivity of serology in patients with erythema migrans, without an associated loss of specificity. We also report on two novel serological tests with synthetic antigens, both with promising diagnostic parameters. Lastly, we present the results of a large clinical study on so-called cellular tests for LB, showing that these tests lack the specificity required for diagnosing LB. In addition, this thesis describes the clinical characteristics of patients with long-lasting symptoms attributed to LB. We found that these symptoms frequently have a profound impact on patients’ lives. We conclude that these patients frequently experience a lack of understanding or validation from both peers and healthcare providers, and we suggest various avenues for future research and improvements to clinical care.