Tendon pathology is frequently sub-clinical prior to frank rupture, denoting the need for non-destructive methods of assessing disease presence and progression. Despite the lack of clinical presentation, previous studies have observed that distinct changes to the tendon microstructure are present, and that such qualitative changes have a dose-response relationship with the level of damage accumulated. These initial findings suggest that there is value in investigating the physical nature of damage within tendon, not only to better understand the pathological process, but also to gain insight into reparative processes and develop treatments. However, a necessary first step towards carrying out these avenues of research is to develop diagnostic tools for quantitatively assessing the level of damage present. In this study, we established a dose-response relationship between a quantitative measure of structural damage and the level of global damage induced. Furthermore, we developed and validated an automated technique for quantifying matrix disorganization (damage), which correlates and co-localizes strongly with manual quantification. In combination, these findings allow us to measure the amount of damage accumulation of a region of tendon on a clinical scale, rapidly and accurately.