A rapid, sensitive and enantioselective method was developed and fully validated for the separation and determination of lansoprazole enantiomers in rat plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analytes and the internal standard (esomeprazole) were both extracted from plasma samples by liquid–liquid extraction with diethyl ether–dichloromethane (70:30; v/v). Satisfactory resolution (Rs = 2.0) was achieved within 7.3 min on a Chiralpak ID column (250 × 4.6 mm, 5 μm) employing acetonitrile–water (60:40, v/v) as the mobile phase at a flow rate of 0.6 mL/min. The acquisition of mass spectrometric data was performed in the multiple reaction monitoring mode coupled with a positive electrospray ionization source. A comprehensive validation of this method was rigorously conducted over the concentration range of 1.00–500.0 ng/mL for both enantiomers. All of the validation data demonstrated that the desirable linearity, sensitivity, accuracy, precision, recovery and stability were attained from the proposed approach. The established method was successfully applied to a stereoselective pharmacokinetic study of lansoprazole enantiomers in rat plasma after oral administration of 3 mg/kg racemic lansoprazole or dexlansoprazole. No chiral inversion was observed during the experimental procedure.