Spinal cord injuries (SCIs) are commonly studied by causing injury to rodent spinal cords in vivo and analyzing histological and behavioral results post injury. However, very few researchers have investigated the deformation of the in vivo spinal cord dynamically during impact. This knowledge would help to define relationships between impact parameters, internal structure deformation (such as grey and white matter), and histological and functional outcomes which could be used to improve SCI treatment and prevention methods. The objective of this study was to develop a method of tracking the real-time internal and surface deformations of an anesthetized rat’s spinal cord during injury. Twelve Sprague Dawley rats were used for this study. After a laminectomy of C5, two radio-opaque beads were injected into the cord (one dorsal, one ventral bead) using a custom technique. Four additional beads were glued to the surface of the cord caudal and cranial to the injection site (one dorsal, one ventral). The dorsal surface of the cord was impacted using a hydraulic actuator at approximately 130mm/s to a depth of 1mm. The spine was imaged laterally at 3,000 fps using a custom high speed x-ray system and the bead motion was tracked in the x-ray video. The internal dorsal bead had significantly larger anterior displacements than the internal ventral bead (0.85 ± 0.14 vs. 0.30mm ± 0.11mm (avg±SD) respectively) and than all surface beads. The internal ventral beads had significantly larger anterior displacements than the ventral surface bead displacements. The dorsal beads had larger maximum velocities in the anterior direction than the ventral beads. The bead migration was not significant in the anterior direction, and was small in the cranial direction (approximately 0.04mm). Harvested spinal cords showed that the internal dorsal beads were in the dorsal white matter. The internal ventral beads were in the ventral white matter for half of the animals and the ventral grey matter for the remaining. There were no significant differences found between white and grey matter motion. These results indicate the merit of this technique for measuring in vivo spinal cord deformation.