Suitable analytical methods to determine the radiochemical purity of validamycin and melamine are not described in the literature. High performance ion-exchange chromatography for the detection of underivatized validamycin and melamine has been proposed. The compounds were separated on HPLC using a styrene-divinylbenzene copolymer based cation-exchanger. For the separation of validamycin a resin in the hydrogen-form has given a good separation and for melamine in the Na+-form. For the first time an ion-exchange separation of validamycin is described allowing the separation of at least four components of commercial validamycin: validamycin A, validamycin B or G, validoxylamine A and validoxylamine B or G without prior derivatization. The methods were applied to determine the radiochemical purity of 14C- or 3H labeled compounds.