The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged organs, including limbs, brain, heart, tail, and others. Studies on axolotl shed light on cellular and molecular pathways ruling progenitor activation and tissue restoration after injury. This knowledge can be applied to facilitate the healing of regeneration-incompetent injuries, such as bone non-union. In the current protocol, the femur osteotomy stabilization using an internal plate fixation system is described. The procedure was adapted for use in aquatic animals (axolotl, Ambystoma mexicanum). ≥20 cm snout-to-tail tip axolotls with fully ossified, mouse-size comparable femurs were used, and special attention was paid to the plate positioning and fixation, as well as to the postoperative care. This surgical technique allows for standardized and stabilized bone fixation and could be useful for direct comparison to axolotl limb regeneration and analogous studies of bone healing across amphibians and mammals.