Abstract: Objective: To establish a high performance liquid chromatography(HPLC)method for the determination of benzalkonium chloride in tobramycin in-situ forming eye gel.Methods: HPLC, the chromatographic column was Kromasil C18(250.0 mm 4.6 mm, 5 m), the mobile phase 0.07 mol /L ammonium acetate solution(including 1% triethylamine, adjusting with glacial acetic acid to pH 5.0)-acetonitrile(70∶ 30).The detection wavelength 215 nm, the column temperature 35 ℃, the flow rate was 0.8 ml /min.Results: The calibration curve was linear in the range of 16.22-81.10 g /ml for benzalkonium chloride(r = 0.999 8,n = 5), the regression equation was Y = 6 091.9X+ 10 114, the average recovery was 99.85%,RSD was 1.07%.Conclusions: The method is convenient, sensitive and accurate with a good repeatability for the determination of benzalkonium chloride in tobramycin in-situ forming eye gel.