To establish a UPLC-ESI-MS method for the assay of UDP-glucuronosyltransferases(UGT) 1A9 activities in human and animal liver microsomes, to investigate the interspecific differences of UGT1A9. The probe substrate of propofol (m/z 177.3), II phase metabolite of propofol glucuronide (PG) (m/z 377.3, [M + Na] + ), and internal standard (IS) of 3-acetamidophenol (m/z 152.2, [M + H] + ) were quantified by selective ion monitor (SIM), using electrospray ionization-MS (ESI-MS), acquity UPLC BEH C 18 column (50 mm × 2.1 mm, 1.7 μm) and a mixture of acetonitrile(B)-0.5% acetic acid(A) as flow phase in gradient elution at flow rate of 0.3 mL·min −1 . The linearity was within 0.2 to 12.8 μg·mL −1 for PG. The intra- and inter-day precisions were less than 3% and the accuracies ranged from −3.46% to 3.03%. The recoveries of PG from human liver microsomes (HLMs) were 96.5%-98.6%. The method was successfully used to determine the kinetics of propofol glucuronidation in human and different animals liver microsomes. The results indicated that the kinetics of propofol O-glucuronidation in liver micro-somes from human, mouse, rabbit and sheep were fitted to the substrate inhibition equation, whereas the kinetics in rat liver micro-somes was fitted to the two-enzyme Michaelis–Menten equation. The intrinsic clearance (CL int ) of propofol glucuronidation in different liver microsomes was in the order of mice > rats (CL int1 + CL int2 )>sheep>rabbits>human. The method is rapid, sensitive and selective for assay of UGT1A9 activities in liver microsomes. The results indicated that the kinetics of propofol O-glucuronidation in rat liver microsomes is different from that in HLMs, and the CL int value for propofol O-glucuronidation in liver microsomes from mice, sheep, and rabbit are remarkably higher than that in HLMs. In animal experiments for drug development, these interspecific differences should be taken into consideration.