When analyzing trace components in vivo, it is important to truly capture the target molecules, and even if advanced separation analysis methods are used, it is necessary to perform sufficient pretreatment. In recent years, chromatography and mass spectrometry have become commonly used for the analysis of biological components. Rapid development in both of these technologies has made it possible to capture previously undocumented phenomena. We have comprehensively analyzed cholesterol metabolites, including bile acids and their conjugated metabolites, in vivo, using both technologies. In addition, we have applied these techniques not only to small molecules in vivo but also to the analysis of biopolymers interacting with them. These results have contributed to the development of drug therapy, diagnosis, and other medical procedures, and we introduce some of these examples in this paper.