Wound healing is a major medical challenge as acute injuries affect millions annually, while chronic wounds such as diabetic foot and venous leg ulcers persist in 1–2% of patients, driving substantial morbidity and ~4% of global health-care expenditure. In response to an injury, restoration of skin barrier is critical process and regulated at multiple levels involving coordinated communications and interaction among keratinocytes, immune cells, and fibroblasts. In vivo mouse models are essential for dissecting the cellular and molecular regulators of normal and pathological repair, especially given the availability of transgenic tools and cell-sorting methods. However, the most common back-punch excisional model heals predominantly by panniculus carnosus-mediated contraction (up to 90%), which limits granulation tissue formation and affects reepithelialization. The mouse tail excisional model provides an alternative to back-punch model as wound closure yielding robust granulation and epithelial coverage due to the “splinting” effect of the tail structure. Here, we describe a detailed, reproducible protocol for tail excisional wounds optimized for histology, cryosectioning, and downstream cell isolation for single-cell multi-omic analyses.