Attenuated total reflection Fourier transform infrared spectroscopic imaging combined with tape-stripping is an advantageous approach to map the depth penetration and lateral distribution of topically applied chemicals in Stratum corneum (SC) and the conformational order of SC lipids. Tape-stripping progressively removes layers of SC, and chemical imaging provides spatially resolved information on the chemical composition of both the newly exposed SC surface and of the tapes used for stripping. The procedure is rapid, minimally invasive, and does not necessitate cross-sectioning of the skin. This approach offers a simple and direct way to determine the distribution of exogenous volatile and non-volatile chemicals in SC as a function of the chemical composition of the formulation and time, and the conformational order of SC lipids in native and topically treated skin. The procedure described here is well suited to address questions of relevance for the areas of drug delivery, dermatology, and skin care.