Fourier transform-second-harmonic generation (FT-SHG) imaging is used to quantitatively assess the structural organization of collagen fibers in tendonitis-induced horse tendons. Fiber orientation, isotropy, and the ratio of forward to backward SHG signal (F/B ratio) are used to differentiate the fiber organization between the normal and diseased horse tendons. Each second-harmonic generation (SHG) image is divided into several smaller regions of interest (ROI) and the aforementioned quantitative metrics are calculated across the whole grid. ROIs are further labeled as dark (no or minimal presence of fibers), isotropic (random fiber organization), or anisotropic (regular fiber organization) regions. Results show that the normal tendon possesses minimal isotropic regions and small standard deviations in the histograms of orientation and F/B ratio, indicating an intact and highly regular fiber organization. However, the tendonitis-induced horse tendons possess higher number of dark and isotropic regions, and larger standard deviations of the measured parameters, suggesting significantly disoriented and disorganized collagen fibers. This type of quantification would be highly beneficial in diagnosing and determining the stage of tendonitis in clinical settings. Not limited to tendonitis, the technique could also be applied to other diseases that structurally affect collagen fibers. The advantage of FT-SHG over the conventional polarization microscopy is also discussed.