One of the most relevant postoperative problems in liver transplantation is the initial graft dysfunction, which is generated by the oxidative stress due to ischemia and/or reperfusion. This leads to ischemia-reperfusion injury of the transplanted organ. This injury can be severe and, when the primary graft is nonfunctional, an urgent retransplantation is required. In this context, tools to monitor the oxidative stress in liver grafts would improve the surgical decision-making for transplantation, increasing its success rate. In this study, we evaluated the potential of time-resolved fluorescence spectroscopy to measure oxidative stress in liver grafts before transplantation. This was performed in livers after ischemia and reperfusion kept in 0 °C (control group) and or 20 °C (ischemia-reperfusion injury group). Both fluorescence spectra and lifetimes were monitored immediately, each 1 hour for the first 12 hours, and at 24 hours after the removal of the liver. The hepatic tissue was excited by lasers emitting in 378 nm and 445 nm for investigation of possible metabolic rates associated to NAD(P)H, FAD, lipopigments, and lipofuscin molecules. The fluorescence decay curves were fitted to the convolution between the instrument response function with a bi-exponential decay for 378 nm and a tri-exponential decay for 445 nm by using the SPCImage software. For both excitation wavelengths, the relative weights for the first exponential component decreased faster as a function of time for the ischemia-reperfusion injury group compared to the control group. This suggests time-resolved spectroscopy is a promising technique to help clinicians to make decisions before liver transplantation.