Objective: To evaluate the detection of near-infrared fluorescence from prostate tumors stained with a prostate-specific membrane antigen (PSMA)-targeted tracer developed in our institution with a novel robotic imaging system. Methods: Prostate cancer cell lines PC3-pip (PSMA positive) and PC3-flu (PSMA negative) were implanted subcutaneously into 6 immunodeficient mice. When tumors reached 5 mm, a PSMA-targeted fluorescent conjugate was injected intravenously. The first 3 mice underwent near-infrared imaging immediately and hourly up to 4 hours after injection to determine the time necessary to obtain peak fluorescence and were killed. The last 3 mice were imaged once preoperatively and were euthanized 120 minutes later. Excision of the tumors was performed by using a novel robotic imaging system to detect near-infrared fluorescence in real time. Specimens were submitted for pathology. Results: In the first 3 mice, we found 120 minutes as the time needed to observe peak fluorescence from the PSMA-positive tumors. We identified discrete near-infrared fluorescence from 2 of 3 PSMA-positive tumors with the robotic imaging system. Surgical margins were negative for all excised specimens except for one PSMA-negative tumor. Conclusions: Real-time near-infrared fluorescence imaging of prostate cancer is feasible with a novel robotic imaging system. Further research is needed to optimize the signal intensity detectable from prostate cancer with our tracer. Toxicologic studies are needed before its clinical use. © 2013 Elsevier Inc.