Lumbar vertebra motion analysis provides objective measurement of lumbar disorder. The automatic tracking algorithm has been applied to Digitalized Video Fluoroscopy (DVF) sequence. This paper proposes a new Auto-Tracking System (ATS) with a guide device and a motion analysis to automatically measure human lumbar motion. Digitalized Video Fluoroscopy (DVF) sequence was obtained during flexion-extension lumbar movement under guide device. An extraction of human vertebral body and its motion tracking were developed by particle filter. The results showed a good repeatability, reliability and robustness. In model test, the maximum fiducial error is 3.7% and the repeatability error is 1.2% in translation and the maximal repeatability error is 2.6% in rotation angle. In this simulation study, we employed a lumbar model to simulate the motion of lumber flexion- extension with the stepping translation of 1.3 mm and rotation angle of 1°. Results showed that the fiducial error was measured as 1.0%, while the repeatability error was 0.7%. The sequence can be detected even noise contamination as more as 0.5 of the density. The result demonstrates that the data from the auto-tracking algorithm shows a strong correlation with the actual measurement and that the Vertebral Auto-Tracking System (VATS) is highly repetitive. In the human lumbar spine evaluation, the study not only shows the reliability of Auto-Tracking Analysis System (ATAS), but also reveals that it is robust and variable in vivo. The VATS is evaluated by the model, the simulated sequence and the human subject. It could be concluded that the developed system could provide a reliable and robust system to detect spinal motion in future medical application.