OBJECTIVE： To establish the method for the determination of related substances in Ornithine aspartate for injection. METHODS： A HILIC B column-HPLC method was used for the determination of related substances in 3 batches of Ornithine aspartate for injection. The determination was performed on ACE HILIC B column with mobile phase consisted of 0.02 mol/L monopotassium phosphate buffer solution （pH 5.0）-acetonitrile （40 ∶ 60， V/V） at the flow rate of 1.3 mL/min. The detection wavelength was set at 205 nm， and column temperature was 30 ℃. Acid， alkali， light， heat， oxidation forced degradation tests were carried out； established method was compared with the method stated in Chinese Pharmacopeia of 2015 edition. RESULTS： Except for light-destroyed samples， compared with undestroyed samples， main peak area of samples destroyed by other conditions was decreased certainly； the impurity was increased； the separation of impurities with main component peaks was higher than 1.5. The linear range of 5 impurities that maleic acid， arginine， fumaric acid， aspartic acid condensate， 3-mino-2-piperidone and main component ornithine aspartate  were 0.077 2-7.72， 0.464-9.28，0.087 6-8.76，0.403- 10.075，0.082 4-8.24， 1.613-80.7 μg/mL （r＝0.999 7-0.999 9）， respectively. The limits of detection were 0.058， 19， 2.5， 17， 0.35 and 24 ng， respectively； the limits of quantitation were 0.19， 63， 8.3， 56， 1.2， 80 ng， respectively. The average recoveries of 5 impurities were 96.3％-102.1％ （RSD＝0.4％-2.0％，n＝9）. Average contents of 5 impurities in 3 batches of samples were 0 （not detected）， 0， 0.026％， 0.011％， 0.019％， respectively. Compared with the method of Chinese Pharmacopeia， established method obtained similar results， but the peak order of each peak was not consistent in 2 times of system suitability tests. CONCLUSIONS： Established HILIC B column- HPLC method is simple and accurate. It is better than the method stated in Chinese Pharmacopeia in durability and reproducibility. It can be used for controlling relevant substance in Ornithine aspartate for injection.