Objective. To find the relationship between gap of screw-bone interface and pedicle screw pullout strength, and to develop a non-destructive test to predict the pullout strength of pedicle screw. Summary of Background Data. Pedicle screw looseness is frequently observed after spinal implantation surgery. The X-ray image and computer tomography are used to diagnose the gap between screw and bone interface. Nevertheless, the correlation between gap of screw-bone interface and screw looseness is not clear. Pullout test of pedicle screw, which is a destructive testing method, is widely used to evaluate the strength of screw-bone interface in vitro. It usually costs large testing sample to predict the progression of screw looseness using this destructive testing method. Materials and Methods. Sawbones and human cadaveric vertebra were used for this in vitro study. The specimens were divided into three groups, “intact”, “1mm gap” and “2mm gap” (n=10 for each group). The pedicle screws (diameter: 6mm, length: 40mm) were inserted. The fatigue loading apparatus was used to apply axial fatigue loading (20~200N, 5Hz) to the head of the screw to create the interface gap. The tensile-compressive testing apparatus was used to find the interface stiffness and pullout strength of pedicle screw. During the stiffness test, loading was applied on screw head at 20~200N magnitude and 1Hz frequency. The loading of impact testing apparatus was 50N, and the vibration frequency of pedicle screw was measured. During the pullout test, the pedicle screw was pullout axially from the specimen at 5 mm/min, and the maximum pullout force was measured. The relationships between interface stiffness, vibration frequency and pullout strength were analyzed. Results. When the gap between pedicle screw and specimens was expanded to 1mm, the interface stiffness, vibration frequency and pullout strength of pedicle screw significantly decreased (p=0.00). The interface stiffness and vibration frequency are moderately (r=0.58, p=0.00) and highly (r=0.75, p=0.00) correlated to the pullout strength, respectively. Conclusions. The pullout strength could be predicted by measuring the interface stiffness and vibration frequency. This method can be useful to predict the progression of screw looseness during fatigue loading.