A rapid, sensitive, and enantioselective method was developed and validated for determination of ornidazole enantiomers in human plasma by liquid chromatography-tandem mass spectrometry. Ornidazole enantiomers were extracted from 100μl of plasma using ethyl acetate. Baseline chiral separation (Rs=2.0) was obtained within 7.5min on a Chiral-AGP column (150mm×4.0mm, 5μm) using an isocratic mobile phase of 10mM ammonium acetate/acetic acid (100/0.01, v/v). Stable isotopically labeled R-(+)-d5-ornidazole and S-(-)-d5-ornidazole were synthesized as internal standards. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode via positive electrospray ionization, using the transitions of m/z 220→128 for ornidazole enantiomers, and m/z 225→128 for d5-ornidazole enantiomers. The method was linear in the concentration range of 0.030-10.0μg/ml for each enantiomer. The lower limit of quantification for each enantiomer was 0.030μg/ml. The relative standard deviation values of intra- and inter-day precision were 1.8-6.2% and 1.5-10.2% for R-(+)-ornidazole and S-(-)-ornidazole, respectively. The relative error values of accuracy ranged from -4.5% to 1.2% for R-(+)-ornidazole and from -5.4% to -0.8% for S-(-)-ornidazole. The validated method was successfully applied to a stereoselective pharmacokinetic study of ornidazole after oral administration of 1000mg racemic ornidazole.