© 2015 SPIE.Background: Acute scrotum is a urologic condition defined by scrotal pain, swelling, and redness of acute onset. Prompt diagnosis and treatment are necessary to preserve testicular viability. The history and clinical symptoms reported are key to diagnosis and proper treatment, but are not always readily obtained in children, in whom common causes of acute scrotum include testicular torsion, torsion of the appendix testis, and epididymitis. These acute conditions have different causal pathology that mandate specific treatment, hence the importance of early and accurate diagnosis. Methods: A novel optical method was developed for rapid evaluation of acute scrotum by measuring and comparing an absolute measure of tissue oxygen saturation index (TSI %) obtained non-invasively from the affected testis and a control site (skin surface of quadriceps muscle), using a wireless continuous wave near infrared spectroscopy device with spatially resolved configuration (SR-NIRS). Results: Five male children with symptoms of acute scrotum were studied using SR-NIRS in addition to standard practices for diagnosis (history, physical examination, lab tests, color Doppler ultrasonography of the testis and surgical exploration when necessary). SR-NIRS successfully differentiated between three conditions by showing a higher TSI% in 2 cases of epididymitis, lower TSI% in 2 cases of testicular torsion and normal TSI% in 1 case of torsion of the appendix testis. Conclusion: This pilot study indicates that SR-NIRS monitoring of (TSI%) in the testes is feasible as a non-invasive, bedside and quick optical method to identify differences in an absolute measure of tissue oxygenation, and that such measurement offers a new adjunct for differential diagnosis of the causal pathology of acute scrotum in children.