Objective: To determine whether performing robot-assisted partial nephrectomy without warm ischemia “off-clamp” results in favorable postoperative renal functional outcomes compared with the on-clamp method. Methods: We conducted a prospective trial of 80 patients who underwent robot-assisted partial nephrectomy. They were randomized in a 1:1 ratio to undergo the procedure with renal artery clamping or without clamping. The groups were compared across demographics, operative information, perioperative outcomes, and postoperative renal function. We assessed renal function by estimated glomerular filtration rate and renal scintigraphy both preoperatively and at 3 months postoperatively. Results: Patients in the on-clamp and off-clamp groups were similar in age, gender, body mass index, comorbidities, clinical tumor size, nephrometry score, and laterality. Off-clamp procedures were lengthier at an average 178.0 minutes vs 156.0 minutes for on-clamp (P = .011). Estimated blood loss, rates of pelvicalyceal repair, postoperative complications, and positive margins were not different. At a median 3-month follow-up, no significant differences were seen in change in postoperative estimated glomerular filtration rate or percent split renal function between both groups. Conclusion: In this prospective study, off-clamp robot-assisted partial nephrectomy resulted in similar perioperative outcomes compared with the on-clamp technique. No benefit was demonstrated in the preservation of renal function. Urologists may safely employ either an on-clamp or off-clamp strategy depending on surgeon preference and patient-specific factors including baseline renal insufficiency, multiple masses, or solitary kidney.