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Skeletal myofibers naturally regenerate after damage; however, impaired muscle function can result in cases when a prominent portion of skeletal muscle mass is lost, for example, following traumatic muscle injury. Volumetric muscle loss can be modeled in mice using a surgical model of muscle ablation to study the pathology of volumetric muscle loss and to test experimental treatments, such as the implantation of acellular scaffolds, which promote de novo myogenesis and angiogenesis. Here we provide step-by-step instructions to perform full-thickness surgical ablation, using biopsy punches, and to remove a large volume of the tibialis anterior muscle of the lower limb in mice. This procedure results in a reduction in muscle mass and limited regeneration capacity; the approach is easy to reproduce and can also be applied to larger animal models. For therapeutic applications, we further explain how to implant bioscaffolds into the ablated muscle site. With adequate training and practice, the surgical procedure can be performed within 30 min.