This study demonstrates the development and validation of a non-aqueous capillary electrophoresis (NACE) method for enantiomeric determination of omeprazole and its metabolite 5-hydroxyomeprazole. Heptakis-(2,3-di-O-methyl-6-O-sulfo)-beta-cyclodextrin (HDMS-beta-CD) was chosen as the chiral selector in an ammonium acetate buffer acidified with formic acid in methanol. Parameters such as CD concentration, concentration of buffer electrolyte, voltage and temperature were studied in order to optimize both the enantioresolution and migration times. An experimental design was utilized for method optimization, using software Modde 5.0. Validation of the developed method showed good linearity, which was tested over a concentration range of 2.5-500 microM. The regression coefficients for S-omeprazole, S-5-hydroxyomeprazole, R-omeprazole and R-5-hydroxyomeprazole were between 0.996 and 0.997. The limits of detection for the four enantiomers were in the range from 45 to 51microM and the limits of quantification were between 149 and 170 microM with UV detection at 301nm. Using a reduced temperature of 16 degrees C gave improved resolution values, reproducibility and also decreased the occurrence of current loss within the capillary. RSD values for peak migration time were calculated to be between 0.41 and 1.48% using an inter-day study.