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Fluorescence recovery after photo-bleaching experiments were performed in human stratum corneum in vitro. Fluorescence multiphoton tomography was used, which allowed the dimensions of the photobleached volume to be at the micron scale and located fully within the lipid phase of the stratum corneum. Analysis of the fluorescence recovery data with simplified mathematical models yielded the diffusion coefficient of small molecular weight organic fluorescent dye Rhodamine B in the stratum corneum lipid phase of about (3–6) × 10−9 cm2 s−1. It was concluded that the presented method can be used for detailed analysis of localised diffusion coefficients in the stratum corneum phases for various fluorescent probes.