A simple and sensitive RP-HPLC method for the determination of omeprazole R-enantiomer (ROME) and omeprazole S-enantiomer (S-OME) in bulk drug samples and pharmaceutical formulations has been developed and validated. The separation of R-OME and S-OME was achieved on a chiral AGP column using UV detector at 301 nm. The mobile phase consisted of 0.025 mol L-1 di sodium hydrogen phosphate/ acetonitrile, (90:10, V/V) (pH 7.0)–. The linear range of detection was found to be 0.01–150 μg/ml (R2=0.9993) and 0.015–152 μg/ml (R2=0.9999) for R-OME and S-OME respectively. The method has been applied successfully for the determination of S-OME in pharmaceutical preparations. The excipients commonly present in formulations did not interfere in the assay of S-OME.