OBJECTIVE： To improve the determination method of related substances in ornithine aspartate raw material. METHODS： HPLC method was adopted. The determination was performed on Phenomenex Luna NH2 column （two series- connected） with mobile phase consisted of 0.02 mol/L potassium dihydrogen phosphate buffer solution （pH 5.6）-acetonitrile （40 ∶ 60， V/V） at the flow rate of 1.0 mL/min. The detection wavelength was set at 205 nm， and column temperature was 35 ℃. The sample size was 20 μL. RESULTS： The linear range of maleic acid， 3-amino-2-piperidinone， succinic acid， malic acid， fumaric acid and β-aspartyl-aspartic acid were 5.398-26.992 μg/mL（r＝0.999 1）， 5.204-26.021 μg/mL（r＝0.999 3）， 5.149-25.749 μg/mL （r＝0.999 6）， 5.174-25.874 μg/mL（r＝0.999 3）， 5.164-25.823 μg/mL（r＝0.999 3）， 5.194-25.973 μg/mL（r＝0.999 5）. The limits of quantitation were 0.22， 0.53， 51.50， 103.50， 0.26 and 8.31 ng； the limits of detection were 0.13， 0.26， 10.30， 20.70， 0.13 and 2.08 ng. RSDs of precision， stability， and reproducibility tests were no more than 2％. The average recoveries were 99.30％-102.31％（RSD＝1.02％，n＝9）， 99.88％-100.89％（RSD＝0.37％，n＝9）， 99.36％-101.53％（RSD＝0.70％，n＝9）， 99.13％-102.65％（RSD＝1.15％，n＝9）， 100.18％-101.45％（RSD＝0.38％，n＝9）， 99.39％-100.81％（RSD＝0.58％，n＝9）. Neither maleic acid， succinic acid and β-aspartyl-aspartic acid were found in 6 batches of samples. The contents of 3-amino-2-piperidinone were 0.006％-0.056％； the contents of malic acid were 0.014％-0.071％； the contents of fumaric acid were 0.000 5％-0.003 4％； the contents of maximal unknown impurity were 0.013％-0.110％； total contents of unknown impurity were 0.013％-0.120％. CONCLUSIONS： The method is simple， precise， stable and reproducible， and can be used for the determination of the related substances in ornithine aspartate raw material.