The permeability enhancement effect of oleic acid (OA) and propylene glycol (PG) as well as their (1:1 v/v) combined mixture was studied using rat skin. The percutaneous drug administration is a challenge and an opportunity for drug delivery. To date, there is limited research that illustrates the mechanism of penetration enhancers and their combinations on the skin. This project aims to explore the skin diffusion and penetration enhancement of PG, OA, and a combination of PG-OA (1:1 v/v) on rat skin and to identify the potential synergistic effect of the two enhancers utilizing Raman spectroscopy. Dissected dorsal skin was treated with either PG or OA or their combination for predetermined time intervals after which the Raman spectra of the treated skin were collected with the enhancer. A spectrum of the wiped and the washed skin were also collected. The skin integrity was tested before and after exposure to PG. The skin histology proved that the skin integrity has been maintained during experiments and the results indicated that OA disrupted rat skin lipid as evident by changes in the lipid peak. The results also showed that PG and OA improved the diffusion of each other and created faster, yet reversible changes of the skin peaks. In conclusion, Raman spectroscopy is a potential tool for ex vivo skin diffusion studies. We also concluded that PG and OA have potential synergistic reversible effect on the skin.