Skin penetration studies of topically applied drugs are a challenging topic in the development of semisolid formulations. The most commonly used methods can be differentiated by their character into ex vivo/in vivo, invasive/non-invasive and offline/in-line measurements. In this study, we compare ex vivo tape stripping, an invasive technique, which is often used, to confocal Raman microspectroscopy (CRM), ex and in vivo, to establish a correlation between those methods. Retinol was used as a model drug, applied in an oil-in-water emulsion, to compare the skin penetration profiles obtained by the different methods.