Five isocratic liquid chromatography (LC) methods have been examined for the separation of amoxicillin and its related substances on C18or C8columns. The United States Pharmacopeia (USP) assay method gave better selectivity. Similar selectivity was obtained not only on C18columns but also on C8and poly(styrene-divinylbenzene) columns. The good selectivity was confirmed by a second laboratory. A resolution test using cefadroxil was developed for the method performance. Based on the USP method, a gradient LC method was developed for the analysis of related substances in amoxicillin. This method has been proposed for the assay and purity control in the amoxicillin monographs of the European Pharmacopoeia and will be further examined in a collaborative study.