In vitro and in vivo laboratory study. To validate a dual fluoroscopic image matching technique for measurement of in vivo spine kinematics. Accurate knowledge of the spinal structural functions is critical to understand the biomechanical factors that affect spinal pathology. Many studies have investigated vertebral motion both in vitro and in vivo. However, determination of in vivo motion of the vertebrae under physiologic loading conditions remains a challenge in biomedical engineering because of the limitations of current technology and the complicated anatomy of the spine. In in vitro validation, an ovine spine was moved to a known distance in a known speed by an MTS machine. The dual fluoroscopic system was used to capture the spine motion and reproduce the moving distance and speed. In in vivo validation, a living subject moved the spine in various positions under weightbearing. The fluoroscopes were used to reproduce the in vivo spine positions 5 times. The standard deviations in translation and orientation of the 5 measurements were used to evaluate the repeatability of technique. The translation positions of the ovine spine could be determined with a mean accuracy less than 0.40 mm for the image matching technique using magnetic resonance image-based vertebral models. The spine speed could be reproduced within an accuracy of 0.2 mm/s. The repeatability of the method in reproducing in vivo human spine 6DOF kinematics was less than 0.3 mm in translation and less than 0.7 degrees in orientation. The image matching technique was accurate and repeatable for noninvasive measurement of spine vertebral motion. The technique could be a useful tool for determination of vertebral positions and orientations before and after surgical treatment of spinal pathology to evaluate and improve the efficacy of the various surgical methods in restoring normal spine function.