Numerous in vivo studies have been conducted to investigate bone regeneration in orthotopic defect models, but a reliably standardized critical-size defect (CSD) model in small animals is still lacking in tissue-engineering research. Utilizing the expertise of trauma surgeons, veterinary surgeons, and engineers, we evaluated the optimal fixation strategy for in vivo application in terms of surgical suitability and conducted biomechanical studies for 3 fixation devices. Fixation strategies were an external fixation device made of polymethylmethacrylate, widely used in animal care; a self-constructed external clamp-fixation device, designed and manufactured using rapid prototyping techniques; and commercially available 1.2-mm titanium plates used in hand surgery. The CSD was 6 mm in size. Biomechanical testing included compression, 4-point bending, and torsion tests. The surgical procedure was optimized in vitro and validated in a clinical setting in athymic rats in vivo. Despite differences in the results of the biomechanical tests, all fixation devices tested proved suitable for the intended purpose. In conclusion, the evaluated model for stabilizing a CSD in a rat's femur can reliably be used for standardized bone regeneration studies in small animals.