Increasing anterior cruciate ligament (ACL) rupture rates are driving the need for new graft materials which undergo testing to characterize material properties and function. The in vitro collagenase assay is routinely used to determine the degradation rate of collagenous materials. At times, it is used to screen new biomaterials on the basis that results reflect breakdown rates in vivo. However, its predictive potential is inconclusive with no guidelines for acceptable in vitro rates of degradation. Reference data from target tissue/s or existing clinical materials are needed to determine appropriate thresholds. From a summary of reported protocols, the most common bench conditions (bacterial collagenase; unloaded samples) were used to evaluate the in vitro degradation of human tendons used as ACL allografts: patellar, semitendinosus, gracilis, Achilles, tibialis anterior and posterior. Tendons were sectioned in equal volumes and exposed to 100 U collagenase for 1, 2, 4 or 8 h. The change in dry weight was analysed using mixed linear regression. All tendon samples demonstrated a significant reduction in mass over time but the patellar tendon degraded significantly faster than all other tendons (P ≤ 0.004). As all tendons used in this study are clinically accepted, this study provides a range of human tendon reference data for comparative assessment of new tendon and ligament biomaterials. However, the more rapid degradation of the patellar tendon, one of the most successful ACL graft materials, also highlights the limitations of common collagenase assay conditions for predicting in vivo performance, particularly in the absence of suitable comparative controls.