A sensitive and high-throughput liquid chromatography-tandem mass spectrometry system was developed and validated for the simultaneous determination of major human hepatic UDP-glucuronyltransferase forms in human liver microsomes. The analytes were detected using a triple-quadrupole mass spectrometer equipped with an electrospray ionization source in the negative ion and selected reaction monitoring modes. The method provided satisfactory linear concentration range, accuracy, precision, and stability. The developed method was successfully applied to the enzyme kinetic study of estradiol-3-O-glucuronidation, 4-methylumbelliferone-O-glucuronidation, propofol-O-glucuronidation, and 3-azido-3-deoxythymidine glucuronidation in human liver microsomes.