Abstract: OBJECTIVE To establish a method of HPLC to determine the limit and content of potential chiral impurities:L-hydroxy phenyl glycine (impurity 1), L-hydroxyphenylglycine methyl ester (impurity 2) and D-hydroxyphenylglycine (impurity 3) in active pharmaceutical substance D-hydroxyphenylglycine methyl ester qualitatively and quantitatively. METHODS A Crownpak CR(-) chiral column (150 mm×4.0 mm, 5 μm) was used, with perchlorate solution (pH 1.7) -methanol (90:10) as the mobile phase with a flow rate at 0.7 mL·min^-1. The detection wavelength was 226 nm, and the temperature of column was 25℃. RESULTS The calibration curve for impurity 1, impurity 2 and impurity 3 were well linear in the range of LOQ concentration-2.400 μg·mL^-1 (r were 1.000 0, 0.999 9 and 0.999 9, respectively). Average recoveries for impurity 1, impurity 2 and impurity 3 were 99.97%, 100.30% and 103.18%, and RSDs were 0.30%, 0.64% and 0.62% (n=9), separately. CONCLUSION The method is highly specific, accurate and convenient, it can be used for the quality control of the chiral impurities 1, impurities 2 and impurities 3 of D-hydroxyphenylglycine methyl ester.