Objective: To establish an HPLC method for determination of relevant substances in aceglutamide injection. Methods: HPLC was performed on a Sepax HP-C18 column with a mobile phase of 0.1% perchloric acid and methanol (99:1) at detection wavelength of 210 nm and column temperature of 25°C. Results: Six relevant substances were well separated The specificity, linearity, limit of quantitation and detection, correction factor, system precision, solution stability and repeatability were conformed to the requirements of 2010 version of Chinese Pharmacopoeia. Conclusion: Aceglutamide can be completely separated with 6 relevant substances in aceglutamide injection by this method.