Non-invasive in vivo human studies have always been of great importance owing to their advantages over the invasive studies. This research work also presents such a non-invasive study dealing with the penetration of two vitamin derivatives: retinyl acetate and alpha-tocopheryl acetate, into the stratum corneum of two groups of study participants categorized based on age as young (average age of 24.1 ± 3.3 years old) and elderly groups (average age of 68 ± 5.8 years old). According to the increase of age it is expected that intrinsic alterations may occur in the stratum corneum. Understanding these alterations is relevant to the knowledge of the differences between stratum corneum as a function of age-dependence thus, leading to the development of new specific products for ageing. The penetration of these derivatives was semi-quantitatively analyzed using confocal Raman spectroscopic technique with 3510 Skin Composition Analyzer (River Diagnostics, Rotterdam, The Netherlands) with a diode laser of 785 nm wavelength and 26 mW power. The analysis was done in the extended fingerprint region (1800 - 400 cm−1) for the stratum corneum region of the skin considering the depths from surface up to 24 μm. The results of this study clearly indicated that these two vitamin derivatives penetrated significantly into the stratum corneum of both study groups and their penetration was mainly affected by the composition of SC and their physico-chemical properties. The penetration profile of the alpha-tocopheryl acetate displays significant statistical difference (p < 0.05) between groups from surface up to 12 μm.