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The physical barrier of the stratum corneum (SC) is provided by corneocytes and the lateral organization of intercellular lipids, which necessarily includes the orthorhombic organization phase. A review of methods used for in vivo non-invasive measurement of skin barrier function has been carried out and it has been shown that all currently used methods are indirect. The most popular method is the measurement of the transepidermal water loss (TEWL), which does not provide information on biophysical parameters responsible for the barrier function of the SC. It has been shown that confocal Raman microspectroscopy is the most suitable non-invasive method to determine the depth profile of the lateral organization of intercellular lipids, i.e. to study the skin barrier function in vivo.