The analysis of kanamycin sulfate by liquid chromatography using a column packed with poly(styrene-divinylbenzene) and pulsed electrochemical detection on a gold electrode is described. A two-step gradient was necessary to obtain a good separation together with a reasonable analysis time of maximum 45 min. The mobile phases consisted of an aqueous solution of 20 g/l or 60 g/l sodium sulfate, 1.3 g/l sodium octanesulfonate and 50 ml/l 0.2 M phosphate buffer pH 3.0. Sodium hydroxide was added post-column. The influence of the different chromatographic parameters on the separation was investigated. A number of commercial samples were analyzed using this method. Besides the previously reported impurities, such as kanamycins B and C, two other impurities were separated, one of which is called kanamycin D. In total, eight components were separated.