Ketoconazole is an antifungal agent, which is the active ingredient in a shampoo primarily used for the treatment of seborrhatic dermatitis (anti-dandruff shampoo). The shampoo also contains imidazolidinylurea as a formaldehyde releasing preservative. The aim of this study was to develop a HPLC system that allows the determination of both ketoconazole and formaldehyde. The finally selected isocratic system consisted of an Interchrom Nucleosil (25034.6 mm, 5 mm) C column 8 and a mobile phase containing acetonitrile–phosphate buffer 0.025 M, pH 4.0, 45/55 (v/v). Ketoconazole could immediately be determined at 250 nm after injection of diluted shampoo. Formaldehyde was measured at 345 nm after derivatisation with a 2,4-dinitrophenylhydrazine solution. At the selected conditions, the other excipients of the shampoo did not interfere in the assays for both substances. Method validation was performed on both assays. Different selectivity towards ketoconazole and formaldehyde was observed when applying other C columns. This fact, however, did not affect the assays of both 8 substances.