The objective of the present work was to develop and validate a rapid analytical method for determination of enantiomeric purity of hydantoin-5-acetic acid. The method was based on pre-column derivatization of hydantoin-5-acetic acid with phenylamine and complete separation of enantiomers has been achieved on a CHI-DMB analytical column (250×4.6 mm) using n-hexane: isopropanol (80:20 v/v) as mobile phase at a flow rate of 1.0 ml min-1 under UV and optical rotation (OR) detection. Detection wavelength was set at 254 nm. The effects of isopropanol, ethanol, column temperature and flow rate of the mobile phase on enantioselectivity and resolution of enantiomers were evaluated. The method was validated with respect to precision, accuracy, linearity, limit of detection (LOD), limit of quantification (LOQ), robustness and stability. The present method is expected to be accurate, stable, rapid and sensitive and can be used for the determination of the enantiomeric purity of hydantoin-5-acetic acid in bulk samples.