To this end, we implemented a translational approach to identify urinary biomarkers for human DILI. By first identifying proteins related to liver injury in urine of mice exposed to the drug of interest, and subsequently searching for the orthologous proteins in human urine, we aim to more efficiently use the limited availability of human urine samples for biomarker assessment. Here, we show carbonic anhydrase 3, superoxide dismutase 1 and calmodulin as potential urinary biomarkers for APAP-induced liver injury in both mouse and human. The present study was designed to identify novel biomarkers in urine for acute DILI by using APAP as model compound. Applying multiple proteomics techniques allowed us to identify twelve proteins related to APAP-induced liver injury. For the first time, we report the presence of CA3, SOD1 and CaM in urine to be related to APAP-induced liver injury, of which CaM had never been linked to liver injury before. Of these proteins, principally SOD1 and CaM closely associated with plasma ALT, as observed by proteomic profiling and antibody-based methods. CA3 fragments showed a good correlation with plasma ALT with proteomic profiling but this could not be confirmed using Western blotting with a specific antibody for the whole protein. However, CA3 as well as SOD1 and CaM were present in human urine samples after APAP intoxication, and are, therefore, proposed as potential urinary biomarkers for APAP-induced liver injury. Urinary CaM concentration was increased in human APAP intoxications and correlated well with plasma APAP concentration, whereas plasma ALT was not increased. This suggests that CaM might be an early marker compared to plasma ALT. Urinary CaM concentration was also elevated in two cases of human DILI caused by drugs other than APAP, indicating that CaM is not specific to APAP-induced liver injury, but rather to acute hepatocellular injury. High doses of APAP caused liver damage as indicated by an increase in plasma ALT and centrilobular hepatic necrosis. Despite the use of inbred mice, our data indicate that the animals showed a differential response to APAP. This is most likely caused by a variation in glutathione stores in individual mice, since our mice were not fasted before APAP administration. The variation in hepatotoxic response allowed us to correlate urinary protein levels to plasma ALT, a conventional biomarker of liver injury. A major advantage of our experimental design was that we could profile proteins in urine collected in a controlled animal study. Urine samples from patients are difficult to profile in search for biomarkers, because they vary in many features. For example, nutritional status, disease condition, and/or use of other drugs may affect the urinary proteome. Using a translational approach, we were able to identify potential biomarkers for APAP-induced liver injury in mice and confirm the presence of these proteins in human urine samples after APAP intoxication and DILI caused by other drugs. In mice, urine was collected during 24 h after APAP administration, and plasma and liver tissue samples at 24 h after exposure. We measured urine at one time point after APAP administration, but still observed a strong association between plasma ALT values and both SOD1 and CaM levels in urine samples. Yet, we could not assess if these potential biomarkers are EX 527 excreted in urine early after the onset of injury. Nevertheless, SOD1 has previously been reported to appear in rat urine as early as 12 h after treatment with CCl4, another known hepatotoxic chemical. A disadvantage of urine collection during 24 h could be that potentially interesting proteins are difficult to detect because of dilution, particularly those excreted shortly after the onset of injury.