A sensitive and specific liquid chromatography–electrospray ionization–mass spectrometry method has been developed and validated for the identification and quantification of ilaprazole enantiomers in human plasma using granisetron as the internal standard. The method involves a simple liquid–liquid extraction. The analytes were separated by isocratic gradient elution on a Chiralcel OZ-RH (5 μm, 150 × 4.6 mm i.d.) column and analyzed in multiple-reaction-monitoring mode with positive electrospray ionization interface using the respective [M + H]⁺ ions, m/z 367.2 → m/z 183.8 for ilaprazole enantiomers, m/z 313.3 → m/z 138.1 for granisetron. The method was validated over the concentration range of 1.229–1,258 μg L⁻¹ for d-ilaprazole and 1.880–1,926 μg L⁻¹ for l-ilaprazole. The lower limit of quantification was 1.229 μg L⁻¹ for d-ilaprazole and 1.880 μg L⁻¹ for l-ilaprazole. The average extraction recovery of d-ilaprazole and l-ilaprazole was 78 and 71%, respectively. Finally, the validated method was applied to a bioequivalence study of ilaprazole in 24 healthy Chinese volunteers.