Liver ischemia is a process characterised by the decrease of oxygen pressure which leads to the production of Reactive Oxygen Species (ROS), Damage Associated Molecular Patterns (DAMPs), cytokines and chemokines. Once the blood flow is re-established, a massive recruitment of neutrophils induces hepatocyte death in a process called Ischemic Reperfusion Injury (IRI). This type of damage is a common cause of morbidity and mortality in surgical operations with prolonged ischemic time. Currently, there is not any available tool to predict IRI damage in real-time. Hyperspectral imaging (HSI) is a non-invasive device able to quantify information in real time from the emitted electromagnetic spectrum of a specific surface. Here we show the ability of HSI to assess and discriminate between ischemic and non-ischemic liver after 15 min of portal vein and hepatic artery ligation. HSI analysis for StO2% and Near Infrared Perfusion Index showed a drastic reduction in both indices, from 42% to 20% and from 43% to 0% respectively. The ischemia detected by the HSI was confirmed by the systemic and local lactate analysis that showed an increase of 50% (from 1.8 to 3.6 mmol/l), and 76% (from 2.3 to 9.7 mmol/l).