A reversed-phase high performance liquid chromatographic method was developed for the determination of amino acids in eptifibatide injection using phenyl isothiocyanate as pro-column derivatization reagent.The chromatographic parameter was optimized,and the analysis of amino acids was performed on a Agilent XDB-C18 column（5 μm,150 mm×4.6 mm）,using 0.1 mol/L sodium acetate（adjusting the pH to 6.0 with acetic acid） and acetonitrile（90∶10,A）-acetonitrile（B） as mobile phase by gradient elution at a flow rate of 1.0 mL/min and an injection volume of 5 μL.The detection wavelength was set at 254 nm,and the column temperature was set at 30 ℃.Under the optimal conditions,the amino acids were effectively separated. good linearities were obtained in the range of 1.23-3.68 mmol/L for all amino acids,with correlation coefficients(r) of 0.999 1-0.999 6.The method showed high selectivity,good repeatability,accuracy and stability,and could be applied in the accurate quantification of amino acids in eptifibatide injection.