In acid solution, idoxuridine (5-iodo-2′-deoxyuridine) is decomposed by light to form 2′-deoxyuridine and material whose U.V. absorption has been destroyed. Idoxuridine is hydrolyzed by heat to form 5-iodouracil and 2-deoxyribose in nearly quantitative yield; a small amount of 2′-deoxyuridine is also formed. A procedure for assay of these mixtures is given. The method involves a chromatographic separation on a partition column with a standing phase of 0.1 N HCl and a moving phase of chloroform plus n -butanol (5 : 1 by volume). That portion of the eluate containing the idoxuridine is analyzed by its U.V. absorption. Data are presented to show the accuracy and precision of the method.