Fourier transform-second harmonic generation (FT-SHG) imaging is used as a technique for evaluating collagenase-induced injury in horse tendons. The differences in collagen fiber organization between normal and injured tendon are quantified. Results indicate that the organization of collagen fibers is regularly oriented in normal tendons and randomly organized in injured tendons. This is further supported through the use of additional metrics, in particular, the number of dark (no/minimal signal) and isotropic (no preferred fiber orientation) regions in the images, and the ratio of forward-to-backward second-harmonic intensity. FT-SHG microscopy is also compared with the conventional polarized light microscopy and is shown to be more sensitive to assessing injured tendons than the latter. Moreover, sample preparation artifacts that affect the quantitative evaluation of collagen fiber organization can be circumvented by using FT-SHG microscopy. The technique has potential as an assessment tool for evaluating the impact of various injuries that affect collagen fiber organization.