Several ex vivo and in vitro skin models are available in the toolbox of dermatological and cosmetic research. Some of them are widely used in drug penetration testing. The excised skins show higher variability, while the in vitro skins provide more reproducible data. The aim of the current study was to compare the chemical composition of different skin models (excised rat skin, human skin and human reconstructed epidermis) by measurement of ceramides, cholesterol, lactate, urea, protein and water at different dephts of the tissues. The second goal was to compile a testing system which includes a skin-on-a-chip diffusion setup and a confocal Raman spectroscopy for testing drug diffusion across the skin barrier and accumulation in the tissue models. A hydrophylic drug caffeine and the P-glycoprotein substrate quinidine were used in the study as a topical cream formulation. The results indicate that although the transdermal diffusion of quinidine is lower, the skin accumulation was similar for the two drugs. The different skin models allowed comparable permeability for both compounds, but chemical composition differed. The human skin was abundant in ceramides and cholesterol, while the reconstructed skin contained less water and more urea and protein. Based on these results it can be concluded that skin-chip and confocal Raman microspectroscopy are suitable for monitoring drug penetration and distribution in different skin layers during and at the end of exposure. Furthermore, the human skin obtained from obese patients is not the most relevant model for skin absorption testing in pharmaceutical research.