Background: Interspinous ligament desmotomy (ISLD) has been shown to improve the comfort of horses diagnosed with overriding dorsal spinous processes (DSP), but its effects on spine mobility are unknown.Objective: To objectively quantify the change in mobility of thoracic vertebrae following ISLD using CT and medical modeling software.Study design: Prospective cadaveric manipulation of seven equine thoracolumbar spines collected from T11-L1.Methods: Spines were collected from T11-L1 with the musculature intact. Flexion and extension phases were achieved with a ratchet device calibrated to 2000N. Bone volume CT scans were performed in resting, flexion, and extension phase preoperatively. Interspinous ligament desmotomy was performed at each intervertebral space (n = 8), and bone volume CT imaging was repeated for each phase. The spinal sections were individually segmented and imported into medical software for kinematic evaluation. T11 of each phase were superimposed, the distance between each dorsal spinous process, the total length of the spine, and the maximal excursion of the first lumbar vertebra along with angular rotational information were recorded.Results: The mean distance between each dorsal spinous process increased by 5.6 ± 4.9 mm, representing a 24 ± 21% increase in mobility following ISLD. L1 dorsoventral excursion increased by 15.3 ± 11.9 mm, craniocaudal motion increased by 6.9 ± 6.5 mm representing a 47 ± 36.5% and 14.5 ± 13.7% increase, respectively. The rotation of L1 about the mediolateral axis increased by 6.5° post-ISLD.Conclusion and Clinical Relevance: ISLD increases dorsoventral, craniocaudal, and rotational motion of the equine spine. The computer modeling methodology used here could be used to evaluate multiplanar spinal kinematics between treatments.