Abstract: OBJECTIVE To develop a new HPLC-ELSD method for the determination of isepamicin sulfate and its injection. METHODS The column was Agilent SB-C₁₈(4.6 mm×250 mm, 5 μm). The mobile phase for the determination of related substances consisted of 0.2 mmol·L^-1 trifluoroacetic acid (A) and methanol (B) with gradient program. The mobile phase for the determination of isepamicin and sulfate was 0.2 mol·L^-1 trifluoroacetic acid. The flow rate was 0.8 mL·min^-1. Detector used was an Alltech 3300 ELSD detector. The drift tube temperature was 50 ℃. RESULTS Good separation of isepamicin from main intermediates could be achieved. The standard curves of isepamicin, sulfate, HAPA-gentamine-B, 3N-isepamicin and gentamine-B were linear in the certain range. The average recovery of isepamicin for the determination of isepamicin sulfate injection was 100.0% (RSD=0.8%, n=9). The average recoveries of HAPA-gentamine-B, 3N-isepamicin and gentamine-B for the determination of related substances were 104.4%, 97.6% and 109.0%, respectively. The detection limits of isepamicin, HAPA-gentamine-B, gentamine-B and 3N-isepamicin were 5.1, 4.5, 5.1 and 5.0 μg·mL^-1, respectively. CONCLUSION The method is simple, sensitive and reproducible, and can be used for quality control of isepamicin sulfate and its injecton.