OBJECTIVE:To establish a method for the simultaneous determination of tetracycline hydrochloride and cortisone acetate in Cortisone tetracycline eye ointment. METHODS:HPLC was performed on the column of Phenomenex C18 and shimaduz GL C18 with mobile phase of 0.01 mol/L Sodium dodecyl sulfate solution(adjusted to pH 2.5 with phosphoric acid)-acetonitrile(60∶40,V/V)at flow rate of 1.0 ml/min,detection wavelength was 254 nm,column temperature was 30 ℃,and the injection volume was 20 μl. RESULTS:The linear range was 11.36-227.18 μg/ml for tetracycline hydrochloride(r=0.999 9)and 11.11-222.21 μg/ml for cortisone acetate(r=0.999 9);RSDs of precision,stability and reproducibility tests were no more than 1.2%;recoveries were 96.89%-100.67%(RSD=1.1%,n=9)and 100.04%-101.02%(RSD=0.3%,n=9),respectively. CONCLUSIONS:The method is simple,accurate and specific,and can accurately determine the contents of tetracycline hydrochloride and cortisone acetate in Corti-sone tetracycline eye ointment.