The penetration barrier of mammalian skin resides in the Stratum corneum (SC). Since the intercellular SC lipid matrix represents the major pathway for drug penetration, this remarkable lamellar structure has increasingly attracted interest during recent years. The present work addressed the issue of elucidating the molecular mode of action of two lipophilic penetration enhancers, oleic acid and isopropyl myristate by using oriented SC lipid model membranes of simplistic composition. In addition, the impact of ceramide NP and an artificial CER[EOS] on the structure of the SC lipid model membranes was studied. Different methodological approaches were applied such as neutron diffraction combined with deuterium labelling for elucidating the membrane nanostructure, DSC and FT Raman spectroscopy for the investigation of the thermotropic phase behaviour of the lipids. The experimental findings were confirmed using additional techniques like 2H NMR spectroscopy and MD simulations.