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Combined ATR-FTIR (attenuated total reflection-Fourier transform infrared) spectroscopy and tape-stripping experiments in vitro on porcine ear skin were used to investigate the spatial distribution of different surfactants in the stratum corneum (SC). To reveal a possible connection between the size of the formed micelles and skin penetration, dynamic light-scattering measurements of the aqueous surfactant solutions were also taken. Compared to an alkyl polyglycoside and sucrose laurate, a deeper skin penetration of the anionic surfactants sodium dodecyl sulfate (SDS) und sodium lauryl ether sulfate (SLES) could be related to a smaller size of the formed micelles. Beside the differences in spatial distribution, a link between the physical presence of anionic surfactants in the SC and a decrease of skin hydration was found. Furthermore, the incorporation of SDS and SLES into the SC, even after a brief, consumer-orientated washing procedure with commercially available hair shampoos, was confirmed.