OBJECTIVE:To establish a method for the simultaneous determination of tobramycin and dexamethasone in Tobra-mycin and dexamethasone eye drop. METHODS:HPLC-column derivatization-double wavelength method was performed on the col-umn of Agilent C18 with mobile phase of 0.25% 3-hydroxymethyl aminomethane-acetonitrile-0.5 mol/L sulfuric acid(40:59:1,V/V/V)at flow rate of 1.0 ml/min,detection wavelength was 365 nm and 220 nm,column temperature was 30 ℃,and the volume in-jection was 20 μl. RESULTS:The linear range was 18.01-180.09 μg/ml for tobramycin(r=0.999 5)and 6.02-60.24 μg/ml for dexa-methasone(r=0.999 8);RSDs of precision,stability and reproducibility tests were lower than 1%;recoveries were 98.65%-100.01%(RSD=0.47%,n=9)and 98.92%-100.20%(RSD=0.50%,n=9),respectively. CONCLUSIONS:The method is simple,time-sav-ing,specific,reproducible,accurate and reliable,and can be used for the quality control of Tobramycin and dexamethasone eye drop.