A significant number of new chemical entities (NCEs) disappear due to cytochrome P450 (CYP)-mediated clinical drug-drug interactions in drug discovery. Therefore, a high throughput assay of CYP activities is necessary in order to evaluate the inhibitory or inducible potencies of CYP isoforms with NCEs in early drug discovery. Here, we developed and validated a high-throughput assay to simultaneously monitor the in vitro activities of 8 CYP isoforms. A cocktail of 9 probe substrates for the 8 major CYPs (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5) was incubated with human liver microsomes. Each substrate-derived metabolite was simultaneously analyzed by multiple reactions monitoring with a single ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) run using stable isotope-labeled internal standards. The ultra-fast UPLC gradient allowed each metabolite to be separated within 1 min, providing quantitative linearity of over 2 orders of magnitude. CYP inhibition by 8 well-known inhibitors was confirmed by comparing single substrates with the substrate cocktail. The inhibition curve profiles and IC₅₀ values for all CYPs in the cocktail substrate were similar to those of single substrates. UPLC-MS/MS using a CYP substrate cocktail is a reliable and robust high-throughput method to accurately assess CYP inhibition potencies of newly developed drugs.