Abstract: A number of therapeutical drugs were reported to undergo metabolic activation by drug-metabolizing enzymes. The bioactivation forms reactive metabolite(s), which readily covalently bind to macromolecules, such as proteins and DNA, and then lead to toxicities. In recent years, screening drug candidates for their tendency to generate reactive metabolites during drug discovery and development process and as well monitoring the bioactivation for post-marketing drugs have become increasingly important. Most reactive metabolites are electrophilic in nature and can react with nucleophiles. In vitro microsomal incubations, small nucleophilic molecules, such as glutathione, cyanide and amines are generally used to trap reactive metabolites. Structural elucidation of these stable adducts are conducted by liquid chromatography-tandem mass spectrometry. In this review, different mass spectrometers including triple quadrupole, ion trap, quadrupole-linear ion trap, and high-resolution mass spectrometer, employed for assessing reactive metabolites are described. The recent advances of different techniques and approaches are also discussed.