Mass spectrometry (MS) has emerged as an invaluable technique with a wide array of applications ranging from forensic, proteomic, metabolomic to clinical studies. With the development of different ionization techniques and mass analyzers, even challenging samples can be analyzed, thereby making mass spectrometry an important analytical tool in the field of characterization of bioactive molecules. Mass spectrometry is the only technique that offers the combination of high sensitivity with structural information. The different ionization techniques allow for the examination of analytes ranging from small metabolites to large macromolecular assemblies. In this chapter, we have demonstrated application of MS in the determination of different classes of bioactive compounds with the special emphasis on peptides, alkaloids and lipids. A case study has been demonstrated for each class of compounds which might be helpful for the early learners in the field of mass spectrometry. And, this shall be of interest to the biochemical, biomedical and pharmaceutical communities, as well as those working in other branches of analytical bioscience.