Abstract: A chiral HPLC method was established for the separation and determination of entecavir opticalisomers.Chiral separation of 5 entecavir opticalisomers was achieved under normal-phase chromatographic mode by the Chiralpak AD-H chiral stationary phase.The optimized chromatographic conditions were a mobile phase consisting of n-hexane-isopropanol-alcohol-diethylamine (70∶6∶24∶0.1) with a flow rate of 0.5 mL/min,and an UV detector at 254 nm.The column temperature was set at 30 °C.The influences of the kinds and proportion of organic solvents,acid and base addictives,flow rate and column temperature on the enantio-separation were studied.Under the chromatographic conditions,5 entecavir opticalisomers can be completely separated from each other and the resolution values were not less than 1.9.The validated method is suitable for the quality control of entecavir.