A direct injection high-performance liquid chromatographic (HPLC) method, with column-switching, for the determination of omeprazole enantiomers in human plasma is described. A restricted access media (RAM) of bovine serum albumin (BSA) octyl column has been used in the first dimension for separation of the analyte from the biological matrix. The omeprazole enantiomers were eluted from the RAM column onto an amylose tris(3,5-dimethylphenylcarbamate) chiral column by the use of a column-switching valve and the enantioseparation was performed using acetonitrile-water (60:40 v/v) as eluent. The analytes were detected by their UV absorbance at 302 nm. The validated method was applied to the analysis of the plasma samples obtained from 10 Brazilian volunteers who received a 40 mg oral dose of racemic omeprazole and was able to quantify the enantiomers of omeprazole in the clinical samples analyzed. The assay was able to determine the cytochrome P450 2C19 phenotype of the subjects participating in this study.