Abstract: OBJECTIVE This review summarizes application of high performance liquid chromatography coupled to a tandem mass spectrometer(HPLC-MS/MS) methodology in drug metabolism and pharmacokinetic screens for a series of new chemical entities during the lead optimization stages. METHODS Relevant literatures were consulted, analysis and summary were made. RESULTS The HPLC-MS/MS methods were widely used to support both in vitro and in vivo experiments and for the qualitative assays, such as metabolite identification. CONCLUSION HPLC-MS/MS is a powerful and indispensable analytical tool in drug metabolism and pharmacokinetic screening.