Studying human skin biology can aid in understanding the pathophysiology of skin diseases and developing novel therapies, including tissue engineering approaches. Among various optical and spectroscopic techniques, Raman micro-spectroscopy stands out. This non-invasive method requires minimal sample preparation and provides molecular-level information on healthy and diseased cell types. The following chapter aims to provide a comprehensive guide to the basic methods of Raman micro-spectroscopy analysis of human epidermal samples from the perspective of their biochemical-molecular relationship, without the need for a deep and specialized knowledge in the field of spectroscopy. We do this based on two distinct examples: (1) Unstained cell subpopulations identified by hierarchical cluster analysis are compared with antibody treated sorted cell populations, and (2) skin cell type-specific molecular features are determined, and the composition of unknown cellular mixtures is revealed.