Adapalene, the active constituent of Differin^®, is a novel potent retinoid (vitamin A analogue) for the topical treatment of acne vulgaris. The clinical usefulness of retinoids is limited by a number of side effects, such as teratogenicity and skin irritation. A method has been developed for simultaneous determination of adapalene and retinol in plasma and tissue in in vivo and in vitro studies for the determination of the pharmacokinetic profile and the influence of adapalene on the endogenous retinol level. The new method was developed by coupling an autosampler to an automated solid-phase extraction unit on-line with a gradient HPLC system using UV and fluorescence detection. The low detection limit (0.25 ng mL⁻¹ for adapalene), the small sample weight (50 mg) and the high degree of automation make this method convenient for analysis of biological samples in animal and human studies.