A rapid method for the simultaneous determination of the in vitro activity of the 10 major human liver UDP-glucuronosyltransferase (UGT) enzymes was developed based on the cocktail approach. Specific substrates were first selected for each UGT: etoposide for UGT1A1, chenodeoxycholic acid for UGT1A3, trifluoperazine for UGT1A4, serotonin for UGT 1A6, isoferulic acid for UGT1A9, codeine for UGT2B4, azidothymidine for UGT2B7, levomedetomidine for UGT2B10, 4-hydroxy-3-methoxymethamphetamine for UGT2B15 and testosterone for UGT2B17. Optimal incubation conditions, including time-based experiments on cocktail metabolism in pooled HLMs that had been performed, were then investigated. A 45-min incubation period was found to be a favorable compromise for all the substrates in the cocktail. Ultra-high pressure liquid chromatography coupled to an electrospray ionization time-of-flight mass spectrometer was used to separate the 10 substrates and their UGT-specific glucuronides in less than 6 min. The ability of the cocktail to highlight the UGT inhibitory potential of xenobiotics was initially proven by using well-known UGT inhibitors (selective and nonselective) and then by relating some of the screening results obtained by using the cocktail approach with morphine glucuronidation (substrate highly glucuronidated by UGT 2B7). All the results were in agreement with both the literature and with the expected effect on morphine glucuronidation.