In this study, microemulsion microstructures, key formulation variables, and their relationship to drug transdermal permeation enhancement were investigated. A microemulsion system with high water soluble capacity was developed, using isopropyl myristate, Labrasol, and Cremophor EL as oil, surfactant, and co-surfactant, respectively. The microstructures of the microemulsions were characterized by a combination of techniques including electrical conductivity measurement (EC), differential scanning calorimetry (DSC), electro-analytical cyclic voltammetry (CV), dynamic light scattering (DLS). Three microemulsion formulations with the model drugs at water contents of 20%, 40%, and 70% representing the microstructures of W/O, Bi-continuous, and O/W were prepared along the water dilution line of oil to surfactant ratio of 1/9. Skin permeation of hydrophobic and hydrophilic model drugs, ketoprofen, lidocaine, and caffeine in the microemulsion formulations was studied using Franz-cells and dermatomed porcine skin. Permeation of all drugs from microemulsions was enhanced significantly compared with the control propylene glycol formulation. The drug permeation flux and the cumulative permeation amount after 24 h increased with water content in the microemulsions, thus correlated to the formulation microstructures of W/O, Bi-continuous, and O/W. The permeation of lipophilic drugs ketoprofen and lidocaine increased with water content in a more pronounced manner, which seemed to follow an exponential growth trend, while the permeation of hydrophilic drug caffeine appeared to increase linearly. Additionally, at the same water content, increasing oil content led to higher ketoprofen permeation.