This paper proposes a new test method based on the toggle effect under transverse loads (cranial–caudal) to investigate the loosening potential of pedicle screw designs. A three-step in vitro testing procedure was developed to mimic the loosening mechanism of pedicle screws. First, the pedicle screw of a certain design is inserted into a bone substitute model specifically designed for the test. Second, a controlled cyclic cranial–caudal loading is applied transversally to the longitudinal axis of the screw for three ascendent load levels (staircase) by a predetermined number of load cycles. Lastly, each pedicle screw is adjusted and subjected to axial pull-out quasi-static testing. The results are used to calculate a loosening index that, together with statistical analysis, indicates the potential for loosening of the specific design evaluated. The proposed testing method effectively provides a simulated environment to evaluate the loosening potential of pedicle screw designs. The proposed loosening index calculation may be used to compare different pedicle screw designs. The proposed methodology was verified as a valuable tool to investigate the influence of the cranial–caudal loads on pedicle screw behavior. It offers a new alternative for use in preclinical studies on the loosening potential of pedicle screw designs.