Objective: To investigate an HPLC method to determine glycine content in human coagulation factor VIII. Methods: The amino bonded column was applied to sep. glycine; the mobile phase consisted of acetonitrile-phosphate buffer (50:50) at a flow rate of 1.0 mL·min-1; the detection wavelength was 205 nm, the column temperature was 40°C, and the injection volume was 20 μL. The content of glycine in human coagulation factor VIII was calculated by area external standard method. Results: In this experiment, the results showed that the separation of glycine and arginine met the requirement, and the linearity was good at the range of 1.02 mg·mL-1 to 5.04 mg·mL-1. The average contents in three batches of samples were 8.70 mg·mL-1, 8.76 mg·mL-1, and 8.80 mg·mL-1; RSD were 0.57%, 0.32% and 0.50%. Conclusion: The results of methodol. validation show that the HPLC method can be used to determine the content of glycine in human coagulation factor VIII.