Here we present a novel, compact 3D-printed diffusion cell as an in vitro tool for skin permeation investigation. As proof-of-concept, a diffusion cell for studying the permeation of a model molecule (FITC-dextran, 4 kDa) through explanted mice skin is fabricated and characterized. Good viability of the tissue up to 24 h incubation in the cell is demonstrated via MTT assays. The real-time diffusion of the molecule by means of fluorescence microscopy allowed the determination of its diffusivity through the skin (~2·10−10 m2/s). Our results open the door for the real-time, high-throughput and cost-effective investigation of skin in any labs.