The anxiolytic agent lorazepam (LZP) and anticonvulsant valproic acid (VPA) are mainly eliminated in humans by glucuronidation. This study aimed to characterize UDP-glucuronosyltransferases responsible for (R,S) lorazepam glucuronidation and quantitatively predict in vivo metabolic drug-drug interaction between LZP and VPA. Formation of the glucuronide metabolites (R-LZPG and S-LZPG) were quantified by HPLC. Recombinant human UGT enzymes were expressed in HEK293 or baculovirusinsect cells. Kinetics of R- and S-LZP glucuronidation by HLMs (n=5) exhibited substrate inhibition. Mean derived binding affinity (Km) and Vmax of R- and S-LZPG kinetics were 36.7 ± 11.4 μM and 7.3 ± 2.1 pmol/min.mg, and 50.3 ± 18.2 μM and 11.7 ± 5.2 pmol/min.mg, respectively. Substrate inhibition with both metabolites was weak (Km 10- to 17-fold lower than Ksi). Twelve recombinant UGTs were screened for LZP glucuronidation activity at LZP concentrations of 10, 50 and 250 μM. UGT 2B4, 2B7 and 2B15 catalyzed S-lorazepam, but the highest activity is observed for UGT2B15. R-lorazepam was catalyzed by UGT 2B4, 2B7 and 2B15. Notably, UGT 1A7 and 1A10 which are expressed in gastrointestinal tract metabolized only R-lorazepam. To identify UGTs responsible for lorazepam glucuronidation in the liver, the kinetic studies of hepatically UGTs which exhibited measurable activities were further investigated. Derived Km or S50 values observed with recombinant UGT 2B4, 2B7 and 2B15 were in the range of 13-46 μM which is comparable to those observed by using HLMs. VPA exhibited non-competitive inhibition on R- and S-lorazepam glucuronidation by HLMs with respective Ki values of 3.9 and 3.2 mM. In conclusion, UGT 2B4, 2B7 and 2B15 are likely to be the major enzymes responsible for human liver microsomal R,S-lorazepam glucuronidation. Based on Ki value, 20% increased of LZP area under the plasma concentration time curve was predicted when coadministered with VPA.