Studying skin composition and interaction with topical substances is important both in dermatology and cosmetoscience. Several techniques are utilized and are under development for these purposes. Skin models are in use with different complexity from 3D bioprinted skins through human reconstructed skin substituents to excised tissues. These models are successfully applied in diffusion cells and skin-on-a-chip devices. In the current study two model drugs (caffeine and quinidine) were investigated as a cream formulation in microfluidic skin-on-a-chip device and by confocal RAMAN spectroscopy. Excised skins and skin substituents were compared in regard of their chemical composition, barrier function and permeability. First the measuring system was optimized and then the properties of the skin models were characterized. Caffeine, as a hydrophilic drug easily penetrates through the skin, the more lipophilic quinidine reached a much lower concentration in the perfusion fluid. The accumulation of the drugs in the upper layers of the epidermis was similar showing that quinidine can well-penetrate to the lipophilic matrix of stratum corneum, but does not cross the full thickness skin barrier as easily. In the penetration of caffeine the transappendageal route has a crucial role, this can explain the two order of magnitude difference in absorption compared to quinidine. Furthermore, quinidine interacts with several transporters in the skin which influences its penetration profile.