Abstract: A HPLC method was developed for the determination of enantiomeric purity of R-rabeprazole sodium. A chiral column (CHIRALPAK-AGP) packed with α1-acid glycoprotein immobilized on silica gel was used as the stationary phase,with a mixed solution of acetonitrile and phosphate buffer (pH 6.0) as the mobile phase for elution. UV detection at the wavelength of 285 nm was utilized. Linear relationship between the peak area and the mass concentration of S-rabeprazole sodium were found in the range of 38.4-576 μg·L-1 with detection limit (3S/N) of 0.2 ng and the lower limit of determination (10S/N) of 0.56 ng. The recovery rates measured by standard addition method were in the range of 91.1%-102%,and RSD were in the range of 2.4%-4.3%.