Thiamylal, a widely used anesthetic drug, has two enantiomers. We developed a novel and simple method for measuring thiamylal enantiomers in human serum using reversed-phase high-performance liquid chromatography. R(+)- and S(-)-Thiamylal were separated using a chiral mobile phase containing beta-cyclodextrin, and detected at the range of 50 ng/ml-25 micrograms/ml in serum. The relative standard deviations of R(+)- and S(-)-thiamylal were 3.4-8.7% and 2.8-8.7% for the intra-day assay, and 2.8-12.0% and 2.8-13.0% for the inter-day assay. This method may be applied to enantioselective pharmacokinetic studies of thiamylal.