Detecting, identifying, and characterizing molecules and structures as the substance of life are essential steps toward unveiling their role as a sole entity or more commonly as a part of a larger molecular assembly for specific biological functionalities. Next, it is of pivotal importance to translate the acquired knowledge of molecular structures and their activity to a more comprehensive understanding of the biological process, such as the biochemical foundations of diseases or metabolic disorders. The aim and ultimate outcomes of this thesis were to address these two aspects by offering new analytical strategies such as focusing on the molecular structure assessment of small molecules, including drug metabolites and potential biomarkers, and drug targets. Addressing challenges encountered in protein characterization and protein interactions studies. And lastly, taking a step forward and analyzing a dynamic chemical reaction in an online fashion by mass, tandem mass, and ion mobility spectrometry.