Abstract: A novel method was developed for the content assay and related substances determination of neomycin sulfate by high performance liquid chromatography combined with pulsed amperometric detection(HPLC-PAD). The HPLC was performed on Thermo Acclaim^TMAmG C₁₈(4. 6 mm×150 mm, 3 μm). The mobile phase consisted of aqueous solution with 2% trifluoroacetic acid containing 0. 01% pentafluoropropionic acid and 0. 6%NaOH. The pulsed amperometric detector was operated with aquadruple-potential waveform for the detection. Neomycin B, Neomycin C and thirteen related substances were adequately separated by the established HPLC conditions. The limits of detection(LOD)and quantification(LOQ)of neomycin B and neomycin C were both 1. 75 ng and 3. 5 ng, respectively. Good linearities of neomycin B and neomycin C were found in their respective ranges which their correlation coefficients were greater than 0. 998 5. The established method is characterized by high specificity, sensitivity and wide range of linearity which has a good application prospect and provides the basis for improving the standard and quality control of neomycin sulfate.