Skin repair is a significant aspect of human health. While the makeup of healthy stratum corneum and epidermis is generally understood, the mobilization of molecular components during skin repair remains largely unknown. In the present work, we utilize multimodal, in situ, mass spectrometry, and immunofluorescence imaging for the characterization of newly formed epidermis, following an initial acute wound for the first 96 h of epithelization. In particular, TOF-SIMS and confirmatory MALDI FT-ICR MS (/MS) analysis permitted the mapping of several lipid classes, including phospholipids, neutral lipids, cholesterol, ceramides, and free fatty acids. Endogenous lipid species were localized in discrete epidermal skin layers, including the stratum corneum (SC), stratum granulosum (SG), stratum basale (SB), and dermis. Experiments revealed that healthy re-epithelializing skin is characterized by diminished cholesterol sulfate signal along the stratum corneum toward the migrating epithelial tongue. The spatial distribution and relative abundances of cholesterol sulfate are reported and correlated with the healing time. The multimodal imaging approach enabled in situ high-confidence chemical mapping based on accurate mass and fragmentation pattern of molecular components. The use of postanalysis immunofluorescence imaging from the same tissue confirmed the localization of endogenous lipid species and Filaggrin and Cav-1 proteins at high spatial resolution (approximately a few microns).