The stratum corneum (SC) water concentration gradient is fundamental to skin's role as a barrier, regulating its physical and biochemical properties. Standard instruments utilizing changes in SC electrical properties to estimate SC water concentration provide simple, rapid measurements but cannot provide true interval data as a function of depth. Confocal Raman spectroscopy (CRS) of human subjects provides non-invasive, real-time, in vivo measures of molecular concentration profiles. A state-of-the-art confocal Raman microspectrometer equipped with a fiber-coupled laser source operating at a wavelength of 671 nm was used to obtain measurements in the high wavenumber region (~2400-4000 cm-1). An aircooled, high-sensitivity back-illuminated, deep-depletion CCD camera captured radiation scattered inelastically from focal planes within the skin in vivo (a high-precision, computer-controlled piezo-electric stage and objective allowing depth resolutions of <5 μm, with over-sampling). High-wavenumber data were analyzed to provide semi-quantitative measures of water concentration ([water] / [protein + water]) across the SC. This new technique was used to study changes in SC water concentration gradients in human skin in vivo, in response to treatment with topical moisturizing products. The results of a blinded, randomized 3-week study in human volunteers will be presented, in particular, the significant, unique effects of a topical moisturizer containing niacinamide on SC water concentration gradient, as measured by CRS, in vivo. The approach to compare SC water gradient effects will be discussed and the utility of this exciting new method will be compared and contrasted to existing methodology.