A high performance liquid chromatography/quadrupole time-of-flight mass spectrometric（HPLC/QTOF-MS） method was adopted to identify the related substances in α-ketophenylalanine calcium(KTP) and a reversed-phase high performance liquid chromatographic（RP/HPLC） method was established for the quantitative determination of its related substances.A C18 column（4.6 mm×150 mm,5 μm） was adopted for quantitative analysis of related substances,with 20 mmol?L-1 potassium dihydrogen phosphate（pH value was adjusted to 3.30 with phosphoric acid） -acetonitrile（85∶15） as mobile phase at a flow rate of 1 mL?min-1,and the detection wavelength was set at 205 nm.The column temperature was 30 ℃ and the injection volume was 10 μL.Two impurities was confirmed as benzoic acid and phenylacetic acid.Under the optimized chromatographic conditions,the calibration curves of the analytes showed good linearities within the mass concentrations of 0.64-5.76 μg?mL-1（r=0.999 6） for benzoic acid and 0.65-5.82 μg?mL-1（r=0999 5） for phenylacetic acid.The detection limits for benzoic acid and phenylacetic acid were 1.3 ng and 1.6 ng,respectively.The average recoveries（n=9） were 99.6% and 99.0% with relative standard deviations（RSDs） of 1.6% and 1.5%,respectively.The results indicated that HPLC/QTOF-MS method could be effectively used to perform the identifications of related substances in α-ketophenylalanine calcium.The method for quantitative determination of related substances was sensitive,reproducible,accurate and reliable.It could be applied in the analysis of the related substances and the quality control of α-ketophenylalanine calcium in raw material.