Background: Intraoperative (IO) image guidance surgery using 3-dimensional fluoroscopic navigation methods, such as the O-arm system, has improved the accuracy of pedicle screw placement in instrumented spine surgery. IO and postoperative (PO) validation of the implant's correct position from radiological images is a decisive step to ensure patient safety and avoidance of complications related to implant misplacement. In this prospective single-center study, the authors investigated the accuracy and agreement of assessment of pedicle screws from IO O-arm images in comparison to PO computed tomography images. This study aimed to determine whether final evaluation of pedicle screws can safely be conducted from IO images that supersedes the PO computed tomography control.Methods: A prospective single-center study was carried out at the Spine Unit in the Department of Orthopedics at Umeå University Hospital between 2019 and 2021. All patients enrolled in the study underwent instrumented thoracolumbar spine surgery using navigation. Imaging data were obtained from IO and PO examinations. Four reviewers-2 attending senior spine surgeons, 1 final year resident in orthopedics, and 1 attending neuroradiologist-classified pedicle screws using the Gertzbein and Robbins classification system. Agreement and accuracy of the reviewers were studied to evaluate the assessment of pedicle screws from IO and PO images.Results: A total of 70 patients (422 screws) were included in the study. There was high accuracy among surgeons both on IO and PO images (0.96-0.97, 95% CI [0.94-0.99] and 0.97, 95% CI [0.94-0.99], respectively), and the overall agreement between all raters was 92% to 98% (95% CI [0.90, 1.00]). The discrepancy in assessment between optimal (Group 1) and suboptimal (Group 2) screws between IO and PO images was as low as 1% to 1.7%, which indicates that very few suboptimal screws are missed in the assessment of IO images.Conclusions: The assessment of navigated pedicle screws using IO images is safe and reliable and may replace the need for further assessment using PO imaging.