Study of skin penetration and distribution of the drug compounds in the skin is a major challenge in the development of topical drug products for treatment of skin diseases. It is crucial to have fast and efficacious screening methods which can provide information concerning the skin penetration and the distribution of the drug molecules in the region of the target. Mass spectrometry imaging (MSI) such as matrix-assisted laser desorption/ionization (MALDI)-MSI offers the opportunity to analyze the drug distribution at micrometer scale, but is a low throughput technique. Cassette dosing of drug molecules has been widely used for two decades as a high throughput screening tool for plasma pharmacokinetic analysis. The purpose of this study is to evaluate the utility of combining MALDI-MSI with cassette dosing to obtain a medium throughput screening technique for drug distribution in the skin directly from thin tissue sections. Excised fresh human skin was treated with two different formulation types containing both single drugs and a cassette with four drugs. Biopsies were taken and analyzed with traditional UHPLC-MS/MS and MALDI-MSI. The results reveal that skin penetration data of the four drugs administered together were in agreement with skin penetration data obtained when the molecules were administered individually. Furthermore, the MALDI-MSI data reveal different distribution profiles of the four drugs which were not possible to deduce from the UHPLC-MS/MS bioanalysis. These findings suggest that combination of MALDI-MSI and cassette dosing can be used as a medium throughput screening tool at an early stage in the drug discovery/development process. Graphical abstract Investigation of drug distribution in human skin explant by MALDI-MSI after cassette dosing.