Acute liver injury (ALI) is a severe disorder resulting from excessive hepatocyte cell death and frequently caused by acetaminophen intoxication. Clinical management of ALI progression is hampered by the dearth of blood biomarkers available. In this study, a bioinformatics workflow was developed to screen Omics databases and identify potential biomarkers for hepatocyte cell death. Then, discovery proteomics was harnessed to select among these candidates those that were specifically detected into the blood of acetaminophen-induced ALI patients. Among these candidates, the isoenzyme alcohol dehydrogenase 1B (ADH1B) was massively leaked into the blood. To evaluate ADH1B, we developed a targeted proteomics assay and quantified ADH1B in serum samples collected at different times from 17 patients admitted for acetaminophen-induced ALI. Serum ADH1B concentrations increased markedly during the acute phase of the disease and dropped to undetectable levels during recovery. In contrast to alanine aminotransferase activity, the rapid drop in circulating ADH1B concentrations was followed by an improvement of the in-ternational normalized ratio (INR) within 10 to 48h and was associated with a favorable outcome. In conclusion, the combination of Omics data exploration and proteomics revealed ADH1B as a new blood biomarker which could be useful to improve the monitoring of acetaminophen-induced ALI.