Near infrared fluorescence capitalizes on the unique features of fluorophores by highlighting critical structures and allowing surgeons to assess vascular perfusion of native tissues. In this review, the authors discuss the physics of fluorescence, characteristics of commonly used fluorophores, and features of approved devices for use in surgery. The authors discuss supporting data and reports of fluorescence guidance in assessment and planning for colorectal and gastroesophageal anastomosis, identification of liver tumors, delineation of biliary anatomy during cholecystectomy, and localization of ureters. Future directions for the use of fluorescence guidance in gastrointestinal surgery include antibody-conjugated fluorophores to detect foci of tumor metastasis.