Prospective single-center study. To compare the accuracy of pedicle screws placed by freehand and under fluoroscopy and robotic assistance with intraoperative image acquisition. Pedicle screws are the most commonly used spinal anchors owing to their ability to stabilize all three spinal columns. Various techniques such as freehand, fluoroscopy-assisted, and navigation-assisted pedicle screw placements have been used with varying degrees of accuracy. Most studies on robotic-assisted pedicle screw placement have utilized preoperatively acquired computed tomography scans. To our knowledge, this is the only study in the literature that compared freehand with fluoroscopy-guided and robotic-assisted pedicle screw insertion with freehand and fluoroscopy. In this prospective study, a total of 1,120 pedicle screws were placed in the freehand group (n=175), 1,250 in the fluoroscopyassisted group (n=172), and 1,225 in the robotic-assisted group (n=180). Surgical parameters and screw accuracy were analyzed between the three groups. The preoperative plan overlapped with the postoperative O-arm scan to determine if the screws were executed as planned. The frequency of clinically acceptable screw placement (Gertzbein-Robbins grades A and B) in the freehand, fluoroscopy-assisted, and robotic-assisted groups were 97.7%, 98.6%, and 99.34%, respectively. With robotic assistance, an experience-neutralizing effect implied that surgeons with varying levels of experience achieved comparable pedicle screw accuracy, blood loss, O-arm time, robot time, and time per screw. No significant difference in these parameters was found between surgeries commencing before and after 2 PM. No significant differences were noted between the planned and executed screw trajectories in the robotic-assisted group irrespective of surgical experience. The third-generation robotic-assisted pedicle screw placement system used in conjunction with intraoperative threedimensional O-arm imaging consistently demonstrates safe and accurate screw placement with an experience-neutralizing effect.