Objective: Ketorolac tromethamine (KT) is considered as a member of Non-steroidal anti-inflammatory drugs that used as an analgesic and antipyretic agent. The main aim of this research was to investigate the effect of some herbal and chemical permeation enhancers on the in vitro skin permeability of KT.Method: Ketorolac permeability experiments through rat skin pre-treated with some of permeation enhancers, namely urea, eucalyptus oil, olive oil, and menthol, were performed in fabricated Franz diffusion cells and compared with hydrated rat skin as control. The permeability parameters evaluated include steady-state flux (Jss), permeability coefficient (Kp), and diffusion coefficient (D). The penetration enhancers permeability enhancement mechanisms were investigated by comparing changes in peak position and their intensities of asymmetric (Asy) and symmetric (Sym) C-H stretching, C=O stretching, C=O stretching (Amide I), and C-N stretching of keratin (Amide II) absorbance using fourier transform infrared spectroscopy (FT-IR), as well as by comparing mean transition temperature (Tm) and their enthalpies (ΔH) using differential scanning calorimetery (DSC). KT permeability parameters through rat skin were evaluated with and without chemical enhancers such as eucalyptus oil, olive oil, menthol, and urea.Result: The skin showed barrier for ketorolac permeability through the whole skin and that diffusion into the skin was the rate-limiting step for drug flux. Urea, eucalyptus oil, olive oil, and menthol were the most effective enhancers as they increased flux 0.02, 0.017, 0.016, and 0.012 times and diffusion coefficient 0.079, 0.194, 0.129, and 1.35 folds in comparison with hydrated skin, respectively.Conclusion: FT-IR and DSC results showed lipid fluidization, extraction, disruption of lipid structure, and irreversible denaturation of proteins in the stratum corneum layer of the skin by permeation enhancers.