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Background: Ex situ machine perfusion facilitates the assessment of livers prior to transplantation. However, currently available markers of liver function poorly predict long‐term graft function. Indocyanine green (ICG) is a liver‐specific dye which, although common in vivo, has never been comprehensively evaluated for the assessment of graft quality during ex situ machine perfusion. This study aimed to assess the utility of ICG in the ex situ setting. Methods: Using a customized long‐term perfusion system, human livers that were not suitable for transplantation were perfused using a red cell‐based perfusate. ICG was delivered into the perfusate on days 0, 1, and 4 to assess ICG clearance (spectrophotometric absorbance at 805 nm) and ICG fluorescence (near‐infrared camera). Results: Sixteen partial livers were perfused for a median duration of 172 h (7.2 days). On day 0, the median ICG perfusate disappearance rate (PDR) was 7.5%/min and the median ICG retention at 15 min was 9.9%. Grafts that survived ≥7 days had a significantly higher median ICG PDR on day 0 (14.5%/min vs. 6.5%/min, p = 0.005) but not on days 1 or 4. ICG perfusion demonstrated that long‐surviving grafts had a significantly lower median red‐value (89.8 vs. 118.6, p = 0.011) and a significantly lower median blue‐value (12.9 vs. 22.6, p = 0.045) than short‐surviving grafts. Conclusion: ICG is a novel marker for the assessment of liver function during ex situ normothermic machine perfusion. ICG PDR and quantitative ICG perfusion can distinguish between long‐ and short‐surviving grafts and demonstrate the utility of ICG in the assessment of graft quality prior to transplant. [ABSTRACT FROM AUTHOR]