Skin forms an excellent barrier against drug permeation, due to the rigid lamellar structure of the stratum corneum (SC) lipids. Poor permeability of drugs can be enhanced through alteration in partition and diffusion coefficients, or concentration gradient of drug with an appropriate choice of solvent system, along with penetration enhancers. The aim of the current investigation was to assess applicability of lipid bilayer alteration by fatty acids and terpenes toward the permeation enhancement of a high-molecular-weight, lipophilic drug, paclitaxel (PCL) through rat skin. From among the fatty acids studied using ethanol/isopropyl myristate (1:1) vehicle, no significant enhancement in flux of PCL was observed (p > 0.05). In the case of cis mono and polyunsaturated fatty acids lag time was found to be similar to control (p > 0.05). This suggests that the permeation of a high-molecular-weight, lipophilic drug may not be enhanced by the alteration of the lipid bilayer, or the main barrier to permeation could lie in lower hydrophilic layers of skin. A significant increase in lag time was observed with trans unsaturated fatty acids unlike the cis isomers, and this was explained on the basis of conformation and preferential partitioning of fatty acids into skin. From among the terpenes, flux of PCL with cineole was significantly different from other studied terpenes and controls, and after treatment with menthol and menthone permeability was found to be reduced. Menthol and menthone cause loosening of the SC lipid bilayer due to breaking of hydrogen bonding between ceramides, resulting in penetration of water into the lipids of the SC lipid bilayer that leads to creation of new aqueous channels and is responsible for increased hydrophilicity of SC. This increased hydrophilicity of the SC bilayer might have resulted in unfavorable conditions for ethanol/isopropyl myristate (1:1) along with PCL to penetrate into skin, therefore permeability was reduced. The findings of this study suggest that the permeation of a high-molecular-weight and lipophilic drug cannot be enhanced through bilayer alteration by penetration enhancers, and alteration in partitioning of drug into skin could be a feasible mode to enhance the permeation of drug.