Background: Fluorescence cholangiography using indocyanine green (ICG) can enhance orientation of bile duct anatomy during laparoscopic cholecystectomy. To ensure clear discrimination between bile ducts and liver, the fluorescence ratio between both should be sufficient. This ratio is influenced by the ICG dose and timing of fluorescence imaging. We first systematically identified all strategies for fluorescence cholangiography. Second, we aimed to optimize the dose of ICG and dosing time in a prospective clinical trial. Methods: PubMed was searched for clinical trials studying fluorescence cholangiography. Furthermore, 28 patients planned to undergo laparoscopic cholecystectomy were divided into 7 groups, receiving different intravenous doses (5 or 10 mg ICG) at different time points (0.5, 2, 4, 6, or 24 hours prior to surgery). Results: The systematic review revealed 27 trials including 1057 patients. The majority of studies used 2.5 mg administered within 1 hour before imaging. Imaging 3 to 24 hours after ICG administration was never studied. The clinical trial demonstrated that the highest bile duct-to-liver ratio was achieved 3 to 7 hours after administration of 5 mg and 5 to 25 hours after administration of 10 mg ICG. Up to 3 hours after administration of 5 mg and up to 5 hours after administration of 10 mg ICG, the liver was equally or more fluorescent than the cystic duct, resulting in a ratio ≤1.0. Conclusion: This study shows for the first time that the interval between ICG administration and intraoperative fluorescence cholangiography should be extended. Administering 5 mg ICG at least 3 hours before imaging is easy to implement in everyday clinical practice and results in bile duct-to-liver ratios >1.0.