A non-invasive ATR-FTIR technique has been used to directly measure skin conditions in vivo. From outside to inside, stratum corneum (SC) consists of fatty layer, one cell layer, a thick layer of lipid and another 10-20 layers of cells. Previous publications [1, 2] have reported the combination of ATR-FTIR with a skin-stripping technique to study human skin in vivo. In this paper, the subtle difference of penetration depth obtained in human skin by using different ATR crystals is discussed. With a Ge crystal (45 degrees), only an alpha-helix protein band was observed in the ATR-FTIR spectrum, which indicated the penetration of light into the one-cell layer of the stratum corneum, because proteins in the cells are in alpha-helix form. In contrast, with a ZnSe crystal (45 degrees), both alpha- and beta-helix protein bands were observed in the FTIR spectrum, which indicated that IR light penetrates into the lipid layer, because the floating lipid contains beta-helix form proteins.