OBJECTIVE To develop a HPLC method for determining the content of related substances in Vitamin C injection. METHODS The sample was analyzed on a NH₂ column (4.6 mm×250 mm，5 μm, Phenomenex) at 45 ℃, with a mobile phase of phosphate buffer (dissolve 6.8 g potassium dihydrogen phosphate in water and dilute to 1 000 mL)-acetonitrile (30∶70), at the detention wavelength of 210 nm and the flow rate was 1.0 mL·min-1. RESULTS The quantitation limit of vitamin C was 45 ng. The peaks of related substances obtained by stress testing were separated thoroughly and antioxidant had no interference with the determination of related substances. CONCLUSION This method was sensitive, special, accurate and suitable for determining the content of related substances in vitamin C injection.