A sensitive, and reliable HPLC method was validated for the simultaneous measurement of omeprazole (OMP) and 5-hydroxyomeprazole (5OH-OMP) in human plasma. OMP, 5OH-OMP, and OPC-18827 (the internal standard) were extracted from human plasma by liquid-liquid extraction into CH2Cl2- iso-PrOH (9:1). Analytes were resolved using a C-18 HPLC column and gradient elution mobile phase containing 50 mM phosphate buffer in acetonitrile (22 - 50% in 43 minutes followed by 15 minutes equilibration). The eluents were monitored by UV detection at 302 nm. The HPLC retention times were 12.19 min for 5OH-OMP, 30.25 min for OPC-18827 and 34.18 min for OMP with resolution factors of 28.3 for 5OH-OMP/ISD and 6.2 for ISD/OMP. Using 0.75 mL of plasma, linearity was established over the range of 20-800 ng/mL for both analytes. Other metabolites were completely separated (OMP-sulfone, 27.50 min) or did not elute under these conditions (OMP- sulfide). In plasma, the analytes were stable at room temperature (bench top, 4 hrs), or refrigerated (57 hrs) and in the autosampler (47 hrs). Sample processing through three freeze/thaw cycles and 2-, 5- and 10-fold dilution with blank plasma before the analysis had no significant effect on the analysis of OMP and 5OH-OMP (>97% recovery). At the lower limit of quantitation, the accuracy was 102.3% for 5OH-OMP and 97.3% for OMP. The method is selective and specific for the analytes in the presence of two other OMP metabolites and endogenous substances.