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The penetration of dexamethasone into human skin ex vivo is reported. X-ray microscopy is used for label-free probing of the drug and quantification of the local drug concentration with a spatial resolution reaching 70 ± 5 nm. This is accomplished by selective probing the dexamethasone by X-ray absorption. Varying the penetration time between 10 min and 1000 min provides detailed information on the penetration process. In addition, the stratum corneum has been damaged by tape-stripping in order to determine the importance of this barrier regarding temporally resolved drug penetration profiles. Dexamethasone concentrations distinctly vary, especially close to the border of the stratum corneum and the viable epidermis, where a local minimum in drug concentration is observed. Furthermore, near the basal membrane the drug concentration strongly drops. High spatial resolution studies along with a de-convolution procedure reveal the spatial distribution of dexamethasone in the interspaces between the corneocytes consisting of stratum corneum lipids. These results on local drug concentrations are interpreted in terms of barriers affecting the drug penetration in human skin.