OBJECTIVE： To improve HPLC method for determining optical isomers in raltitrexed raw material. METHODS： After improved， the column was CHIRALPAK AD-H with mobile phase consisted of n-hexane-anhydrous ethanol-trifluoroacetate （70 ∶ 30 ∶ 0.1，V/V/V）. The sample size was 100 μL. The flow rate of 1.0 mL/min. The detection wavelength was set at 226 nm， and column temperature was 35 ℃. RESULTS： The linear range of optical isomers was 99.68-398.7 ng（r＝0.999 9）. The quantitation limit was 4.945 ng， and detection limit was 1.648 ng. RSDs of precision， stability and reproducibility tests were all lower than 1.0％； the recoveries ranged 99.43％-100.14％（RSD＝0.25％，n＝9）. CONCLUSIONS： The optimized method is simple， accurate， sensitive and reproducible， which can be used for the determination of optical isomers in raltitrexed raw material.