Abstract: AIM To establish an HPLC assay to determine directly, the propranolol glucuronides in microsomal incubate without hydrolysis to their parent enantiomers. METHODS The standard for this direct assay was prepared by incubation of liver microsomes with propranolol. The glucuronide obtained was purified and concentrated with solid-phase extraction and the concentration was measured by an indirect method, i.e. HPLC assay of the propranolol after enzymatic hydrolysis with β-glucuronidase. The direct assay involved separation by HPLC using a C₁₈ reversed phase column, with UV detection at 290 nm. RESULTS The recovery of the assay was 99.4%±1.0% (n=15), the reproducibility of the assay was less than 3% (RSD) for inter-day and 5% (RSD) for intra-day. The standard curves showed excellent linearity over the range 0.24-73.17 μmol·L^-1. Followed Michaelis Menten kinetics, the K_m and C_max for the two enantiomers were very different. The R-enantiomer was glucuronidated at a more efficient rate than its enantiomorph, and was a better substrate. CONCLUSION The sensitive, reproducible and accurate HPLC method was developed to measure directly the propranolol glucuronides and has been applied to determine the stereoselectivity of glucuronidation metabolism of racemic propranolol in rat liver microsomes.