Abstract: A direct method of HPLC without precolumn derivatization was proposed for determination of D-serine. Chromatographic column of Daicel CROWNPAK CR(+) (4 mm×150 mm) and mobile phase of pH 1.0 HClO4 solution were used in the separation, with column temperature of 0 ℃ and flow-rate of 0.3 mL·min-1. 170U UV-detector with wavelength of 200 nm was adopted in the detection. Under the given conditions, the D-serine and its main impurity L-serine were completely separated from each other within 10 min. The resolution factor (Rs) attained to a value of 2.09. Linearity of D-serine was found in the range from 1.00 to 10.00 g·L-1. Detection limit (3S/N) found for the method was 0.20 g·L-1. Tests for recovery and precision were made by standard addition method, values of recovery and RSD′s (n=6) obtained were in the ranges of 93.4%-95.7% and 3.2%-3.6% respectively.