Cutaneous wound healing is an intricate and multifaceted process. Despite these complexities, the distinct phases of wound healing provide a unique opportunity to evaluate the roles of different targets in these coordinated responses. This protocol details an in vivo wound healing assay to study the intersection of cellular, molecular, and systemic effector pathways. The role of certain proteins in the wound healing process can be efficiently explored in vivo through the generation of tissue-specific deficient mice. This approach, although optimized for use with animal models displaying epithelial deficiencies, can be used for other tissue-specific deficiencies, and utilizes simple and cost-effective methods, allowing investigators to precisely devise their experimental design. The coordination of immunological, epithelial, vascular, and microenvironmental factors in wound healing makes this technique a valuable tool for investigators across fields.