The use of fluorescence detection for surgical applications has grown significantly over the last two decades through advances in fluorescent markers, instrumentation and data processing methods. Synergistic combinations of these developments have produced optical methods that provide biochemical, structural and functional information during surgical procedures. In this review, we focus on the surgical methods used clinically for in vivo fluorescence detection. An introduction to basic principles, including the characteristics of fluorescent molecules and intrinsic tissue optical properties, is provided along with an overview of the instrumentation being deployed in the operating room (OR). The intraoperative fluorescent contrast mechanisms currently available in the OR are presented. Discussion is followed by a detailed organ-system summary of clinical research involving in vivo fluorescence detection during surgery. While the emphasis is on clinical applications, a growing body of pre-clinical studies is leading to the discovery of more sensitive and specific molecular fluorescent makers of disease as well as the development of cutting-edge optical technology poised to make its way into the clinical setting.