The epidermis is one of the largest tissues in the human body, serving as a protective barrier. The basal layer of the epidermis, which consists of epithelial stem cells and transient amplifying progenitors, represents its proliferative compartment. As keratinocytes migrate from the basal layer to the skin surface, they exit the cell cycle and initiate terminal differentiation, ultimately generating the suprabasal epidermal layers. A deeper understanding of the molecular mechanisms and pathways driving keratinocytes’ organization and regeneration is essential for successful therapeutic approaches. Single-cell techniques are valuable tools for studying molecular heterogeneity. The high-resolution characterization obtained with these technologies has identified disease-specific drivers and new therapeutic targets, further promoting the advancement of personalized therapies. This review summarizes the latest findings on the transcriptomic and epigenetic profiling of human epidermal cells, analyzed from human biopsy or after in vitro cultivation, focusing on physiological, wound healing, and inflammatory skin conditions.