An isocratic liquid chromatographic method with pulsed electrochemical detection is described for the determination of neomycin in the presence of its impurities. The mobile phase is composed of an aqueous solution containing 35 g/l of sodium sulphate, 1 g/l of sodium 1-octanesulfonate, 14 ml/l of tetrahydrofuran (THF) and 50 ml/l of 0.2 M phosphate buffer pH 3.0. Sodium hydroxide was added post column to enhance the detection. An investigation of different reversed-phase columns indicated that the Discovery (C18 5 microm, 250 mm x 4.6 mm I.D.) column was the most suitable. The proposed method shows high efficiency, allowing the separation of the main component neomycin B from neomycin C and 15 other impurities. A central composite design was used to assess the robustness of the method. The method showed good selectivity, repeatability, linearity and sensitivity. This method was applied to analyse commercial samples.