A method for the determination of optical isomers in tirofiban hydrochloride injection was established.The chromatog. column was CHIRALPAK®IC, and the mobile phase consisted of n-hexane-isopropanol- methanol-trifluoroacetic acid-anhydrous ethylenediamine (40:40:20:0.3:0.3).The detection wavelength was 227 nm, and the flow rate was 0.8mL·min-1.The preparation method of the sample was studied, sample treatment method, diluent ratio and ultrasonic time were screened, it was determined to use rotary evaporation to remove water and then diluent extraction to prepare the test solutionThe method has good specificity, linearity, sensitivity and durability, and is suitable for the determination of optical isomers in tirofiban hydrochloride injection.