Dithranol is one of the mainstays in the topical treatment of psoriasis but its unpleasant side effects result in poor patient compliance and there is an on-going demand for novel formulations, with better absorption and dermal availability characteristics. In the developmental stage, such novel formulations are currently evaluated in artificial membranes, animal models and human subjects. Simple, preliminary in vitro cell permeation studies, performed in the initial stages, will serve to reduce the cost, time and number of animals used in the development of new dithranol formulations. A simple and sensitive isocratic reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been optimized and validated for the determination of dithranol. Samples were analyzed on a C18 column with acetonitrile-distilled water-acetic acid (58:37:5, v/v/v) as the mobile phase, with a flow rate of 1.1 ml/min and UV detection at 394 nm. The assay method was found to be linear from 0.098 to 200 μg/ml, with a mean correlation coefficient (r2) of 0.9986 (n=3). All the validation parameters were within the acceptance range. The validated method was then successfully applied to the analysis of the cellular uptake of dithranol in an in vitro study.