The fabrication of decellularized small intestinal submucosa (dSIS) requires a precise balance between effective cellular removal and the preservation of structural integrity. In this study, we compared four published dSIS protocols representing detergent-based, chaotropic-salt-based, and sequential ionic/alkaline–acidic strategies. Their performance was evaluated based on residual DNA, collagen preservation, surface ultrastructure, and mechanical properties. The best decellularization protocol demonstrated the lowest residual DNA levels, together with better collagen retention, scaffold architecture, and mechanical performance than the other methods tested. The selected decellularized scaffold was used in a murine acute wound model and showed good biocompatibility and integration with the surrounding tissue at 10 days after implantation. However, further extensive testing in murine models is essential before future scaling. Finally, this comparative study provides a practical framework for selecting dSIS preparation methods for skin repair applications.