Aim: To establish a HPLC method for determination of related substance of VB12 in Naipuwei Eyedrop.Methods: The chromatog. column utilized octadecyl silane-bonded silica as filler, and mobile phase was buffer (obtained by adding diammonium hydrogen phosphate 11.5 g in water 1000 mL, adding phosphoric acid 1 mL, and shaking uniformly)-acetonitrile (9:1), and the buffer-acetonitrile (6:4).Flow velocity was 1.2 mL/min, and detection wavelength was 361 nm.The sample was 50 μL.Results: Related substances of VB12 damaged by lighting, oxidation, acid, alkali, and high temperature can be separatedThe detection limit of VB12 was 0.5 ng.The method can effectively detect the related substances of VB12 in Naipuwei Eyedrop.Conclusion: The method has strong specificity, simple operation, high sensitivity, and can be used for controlling the related substances of VB12 in Naipuwei Eyedrop.