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Abstract: Introduction: The PINPOINT® Endoscopic Fluorescence Imaging System (Novadaq, Mississauga, Canada) allows surgeons to visualize the bile ducts during laparoscopic cholecystectomy. Surgeons can continue operation while confirming the bile ducts’ fluorescence with a bright‐field/color image. However, strong fluorescence of the liver can interfere with the surgery. Here, we investigated the optimal timing of indocyanine green administration to allow fluorescent cholangiography to be performed without interference from the liver fluorescence. Methods: A total of 72 patients who underwent laparoscopic cholecystectomy were included in this study. The timing of indocyanine green administration was set immediately before surgery and at 3, 6, 9, 12, 15, 18, and 24 h before surgery. The luminance intensity ratios of gallbladder/liver, cystic duct/liver, and common bile duct/liver were measured using the ImageJ software (National Institutes of Health, Bethesda, USA). Visibility of the gallbladder and bile ducts was classified into three categories (grades A, B, and C) based on the degree of visibility in contrast to the liver. Results: The luminance intensity ratio for the gallbladder/liver, cystic duct/liver, and common bile duct/liver was ≥1 in the 15‐, 18‐, and 24‐h groups. The proportion of cases in which evaluators classified the visibility of the gallbladder and bile ducts as grade A (best visibility) reached a peak in the 15‐h group and decreased thereafter. Conclusions: In the present study, the optimal timing of indocyanine green administration for fluorescent cholangiography during laparoscopic cholecystectomy using the PINPOINT Endoscopic Fluorescence Imaging System was 15 h before surgery. [ABSTRACT FROM AUTHOR]