The combination of ultra-high-performance liquid chromatography and mass spectrometry (UHPLC-MS) offers advantages of high speed, resolution and sensitivity. With these technological advantages, UHPLC-MS has been widely applied to areas of bioanalysis, one of which involves stages of drug discovery and development. This article aims to provide a comprehensive, up-to-date overview of published literature on UHPLC-MS strategies and applications in bioanalyses related to pharmacokinetics and drug metabolism, to give the readers from academia and industry sufficient information about these aspects.