Content of netilmicin was determined by using high performance liquid chromatog. The anal. column was Diamonsil-C18, the mobile phase was 1.5% potassium dihydrogen phosphate (pH 2.5 adjusted by phosphoric acid)-acetonitrile (500:100) with 1.0 mL/min of flow rate, and the detective wavelength was 205 nm. External standard method was used. The standard curve of netilmicin sulfate was linear in the concentration range of 0.1-1.6 mg/mL (r = 0.9998), and the average recovery was 99.5%, RSD = 0.4% (n = 9). High performance liquid chromatog. is a simple, rapid and accurate method for determination of the content of netilmicin sulfate injection.