Fluorescent image-guided surgery has the potential to revolutionize surgery by providing direct tissue visualization using compact imaging systems and targeted contrast agents. Nerve targeting contrast agents are currently under development, where clinical use would decrease morbidity and benefit post-surgical outcomes for a variety of surgical procedures. Herein, we have applied a previously optimized direct administration methodology to resected human prostate specimens to validate the human nerve cross-reactivity of the nerve-specific fluorophore Oxazine 4. Nerves were clearly identified on the posterior lateral surfaces of stained prostate specimens, consistent with prostate neuroanatomy. Additionally, nerve signal-To-background ratios were consistent with the relevant postperfusion murine nerve models, suggesting that contrast levels will match results in murine models when Oxazine nerve-specific fluorophore is applied to human nerves in vivo. The positive human nerve staining demonstrated herein on resected prostate specimens using the optimized direct administration methodology provides a human nerve cross reactivity screening tool for future translational studies of near-infrared nerve specific contrast agents.