In recent years, patient-specific spinal drill guides (3DPGs) have gained widespread popularity. Several studies have shown that the accuracy of screw insertion with these guides is superior to that obtained using the freehand insertion technique, but there are no studies that make a comparison with computer-assisted surgery (CAS). The aim of this study was to determine whether the accuracy of insertion of spinal screws using 3DPGs is non-inferior to insertion via CAS. A randomized controlled split-spine study was performed in which 3DPG and CAS were randomly assigned to the left or right sides of the spines of patients undergoing fixation surgery. The 3D measured accuracy of screw insertion was the primary study outcome parameter. Sixty screws inserted in 10 patients who completed the study protocol were used for the non-inferiority analysis. The non-inferiority of 3DPG was demonstrated for entry-point accuracy, as the upper margin of the 95% CI (−1.01 mm–0.49 mm) for the difference between the means did not cross the predetermined non-inferiority margin of 1 mm (p < 0.05). We also demonstrated non-inferiority of 3D angular accuracy (p < 0.05), with a 95% CI for the true difference of −2.30◦–1.35◦, not crossing the predetermined non-inferiority margin of 3◦ (p < 0.05). The results of this randomized controlled trial (RCT) showed that 3DPGs provide a non-inferior alternative to CAS in terms of screw insertion accuracy and have considerable potential as a navigational technique in spinal fixation.