This study was performed to prospectively compare the clinical and radiographic outcomes between robot-assisted minimally invasive transforaminal lumbar interbody fusion (RA MIS-TLIF) and fluoroscopy-assisted minimally invasive transforaminal lumbar interbody fusion (FA MIS-TLIF) in patients with degenerative lumbar spinal diseases. One hundred and twenty-three patients with lumbar degenerative diseases (lumbar spinal stenosis with instability and spondylolisthesis [degenerative spondylolisthesis or isthmic spondylolisthesis]) who underwent MIS-TLIF in our hospital were included in this study. Sixty-one patients underwent RA MIS-TLIF (Group A) and 62 patients underwent FA MIS-TLIF (Group B). Group A was further divided into Subgroup AI (46 single-level procedures) and Subgroup AII (15 double-level procedures). Group B was further divided into Subgroup BI (45 single-level procedures) and Subgroup BII (17 double-level procedures). The clinical outcome parameters were the visual analog scale (VAS) score, Oswestry Disability Index (ODI) score, operative time, number of intraoperative fluoroscopies, blood loss, postoperative hospital stay, and postoperative complications. The radiographic change measures were the accuracy of screw placement, facet joint violation (FJV), fusion status, and change in disc height at the proximal adjacent segment at the 2-year follow-up. There were no significant differences in the VAS and ODI scores, blood loss, or postoperative hospital stay between Groups A and B (p > 0.05). The operative time was longer in Group A than B (p = 0.018). The operative time was longer in Subgroup AI than BI (p = 0.001). However, there was no significant difference between Subgroups AII and BII (p > 0.05). There was no significant difference in the number of intraoperative fluoroscopies for patients between Groups A and B (p > 0.05). Although the number of intraoperative fluoroscopies for patients was significantly higher in Subgroup AI than BI (p = 0.019), there was no significant difference between Subgroups AII and BII (p > 0.05). The number of intraoperative fluoroscopies for the surgeon was significantly lower in Group A than B (p < 0.001). For surgeons, the difference in the average number of intraoperative fluoroscopies between Subgroups AI and AII was 2.98, but that between Subgroups BI and BII was 10.73. In Group A, three guide pins exhibited drift and one patient developed a lateral wall violation by a pedicle screw. One pedicle screw perforated the anterior wall of the vertebral body and another caused an inner wall violation in Group B. The rate of a perfect screw position (grade A) was higher in Group A than B (p < 0.001). However, there was no significant difference in the proportion of clinically acceptable screws (grades A and B) between the two groups. The mean FJV grade was significantly higher in Group B than A (p < 0.001). During at 2-year postoperative follow-up, there was no significant difference in the fusion status between the two groups (p > 0.05); however, the decrease in disc height at the proximal adjacent segment was significantly less in Group A than B (p < 0.001). Robot-assisted percutaneous pedicle screw placement is a safer and more accurate alternative to conventional freehand fluoroscopy-assisted percutaneous pedicle screw insertion in MIS-TLIF.