The objective of this study was to design and test a protocol for the validation of banking methodologies for arterial allografts. A series of in vitro biomechanical and biological assessments were derived, and applied to paired fresh and banked femoral arteries. The ultimate tensile stress and strain, suture pullout stress and strain, expansion/rupture under hydrostatic pressure, histological structure and biocompatibility properties of disinfected and cryopreserved femoral arteries were compared to those of fresh controls. No significant differences were detected in any of the test criteria. This validation protocol provides an effective means of testing and validating banking protocols for arterial allografts.