Fluorescence molecular endoscopy (FME) is an emerging technique in the field of gastroenterology that holds potential to improve diagnosis and guide therapy, by serving as a 'red-flag' endoscopic imaging technique. Here, we investigated the safety, feasibility and optimal method of administration of EMI-137, targeting c-Met, during FME in Barrett's Esophagus (BE) and report several outcome parameters for early phase FME studies. Methods: FME was performed in 15 Barrett's neoplasia patients. EMI-137 was administered to three cohorts of five patients: 0.13 mg/kg intravenously (IV); 0.09 mg/kg IV or topically at a dose of 200 μg/cm BE (n=1) or 100 μg/cm BE (n=4). Fluorescence was visualized in vivo, quantified in vivo using multi-diameter single-fiber reflectance, single-fiber fluorescence (MDSFR/SFF) spectroscopy and correlated to histopathology and immunohistochemistry. EMI-137 localization was assessed using fluorescence microscopy. Results: FME using different IV and topical doses of EMI-137 appeared to be safe and correctly identified 16/18 lesions, although modest target-to-background ratios were observed (median range of 1.12-1.50). C-Met overexpression varied between lesions, while physiological expression in the stomach-type epithelium was observed. Microscopically, EMI-137 accumulated around the neoplastic cell membranes. We identified several outcome parameters important for the validation of EMI-137 for FME: 1) the optimal administration route; 2) optimal dose and safety; 3) in vivo FME contrast; 4) quantification of intrinsic fluorescence; 5) ex vivo correlation of fluorescence, histopathology and target expression; and 6) microscopic tracer distribution. Conclusions: C-Met targeted FME using EMI-137 may not be the ideal combination to improve BE surveillance endoscopies, however the identified outcome parameters may serve as a valuable guidance for designing and performing future early phase clinical FME studies, independent of which fluorescent tracer is investigated.