Raman spectroscopy was used to detect low quantities of Vismodegib in the skin after its topical application via transfersomes. Vismodegib is a novel antineoplastic drug approved for oral administration for treatment of basal cell carcinoma. Transfersomes loaded with Vismodegib were prepared by thin film resuspension and extrusion, and were characterized physicochemically. Transfersomes were applied to human and pig skin specimens using the Saarbrücken penetration model. The skin was then sectioned by tape stripping, followed by penetration assessment by UV-Vis spectroscopy and Raman spectroscopy in a confocal Raman microscope. Raman signals from Vismodegib and transfersomes were recovered from skin sections, showing a similar distribution in the stratum corneum obtained by the other techniques. On the other hand, pig and human skin showed differences in their penetration profiles, proving their lack of equivalence for assessing the performance of these transfersomes. Raman spectroscopy appears as a potential non-invasive, direct tool for monitoring hard-to-detect molecules in a complex environment such as the skin.