An HPLC method was established to determine the enantiomeric impurities in N-Cbz-L-histidine(Cbz-L-His-OH) and 1-[(1, 1-dimethylethoxy)carbonyl]-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-histidine[Fmoc-L-His(Boc)-OH].An DAICEL CHIRALPAK∼?ZWIX(+) column was used, with the mobile phase of methanol:water:formic acid:diethylamine (900:100:1.9:4.16).The column temperature was 35 °C, the flow rate was 0.2 mL/min, the injection volume was 10 μl, and the detection wavelength was 220 nm.The results showed that the L-and D-enantiomers of Cbz-His-OH as well as Fmoc-His(Boc)-OH could be separated effectively.The detection and quantification limits of Cbz-D-His-OH were 141 and 476 ng/mL, for Fmoc-D-His(Boc)-OH were were 50 and 163 ng/mL, resp.It was linear for the two pairs of enantiomers in the corresponding concentration ranges.The recoveries of both Cbz-D-His-OH and Fmoc-D-His(Boc)-OH were between 90.0% and 100.0%, and the RSDs(n=9) were less than 3.0%.The measurement results showed that contents of enantiomeric impurities in Cbz-L-His-OH and Fmoc-L-His(Boc)-OH were both less than 0.01%.The established method had strong specificity, good reproducibility and high accuracy, which provided a reference for the determination of enantiomeric impurities in Cbz-L-His-OH and Fmoc-L-His(Boc)-OH.