Infrared (IR) spectroscopy has been successfully applied to study the permeation of substances through human skin in a high-throughput manner. The sample of skin was placed on the measuring surface of an attenuated total reflection (ATR) crystal and was divided into several areas. These areas were separated using a specially designed grid created on the surface of the skin and each area was subjected to a different combination of permeant and enhancer. ATR Fourier transform infrared (FT-IR) imaging was applied to measure the permeation of 12 liquid samples through a piece of skin smaller than 5 cm². This work demonstrated that, using the ATR-FT-IR imaging method, it is possible to measure and directly compare the transdermal processes of several permeants under identical conditions.