Abstract: OBJECTIVE To establish a method for determination of related substances in Lincomycin Hydrochloride Eye Drops by HPLC. METHODS The determination was performed on Phenomenex Luna C₁₈ column(4.6 mm×250 mm, 5 μm), and the mobile phase consisted of 77 mL buffer(dissolved 34 g phosphoric acid in 900 mL water and adjust to pH 6.1 with concentrated ammonia and diluted to 1 000 mL with water), 8 mL methanol and 15 mL acetonitrile at the flow rate of 1.0 mL·min^-1. The detection wavelength was 210 nm. The column temperature was 50 ℃. RESULTS The linearity was found in the range of 24.68-370.28 μg·mL^-1 for lincomycin(r=1.000 0). The detection limit of lincomycin was 1.05 ng. CONCLUSION The method can separate lincomycin effectively from its degradation product and antimicrobial, and is suitable for the determination of impurities in Lincomycin Hydrochloride Eye Drops.