Acute kidney injury (AKI) is an important cause of death among hospitalized patients. The 2 most common causes of AKI are acute tubular necrosis (ATN) and prerenal azotemia (PRA). Appropriate diagnosis of the disease is important but often difficult. We analyzed urine proteins by 2-dimensional gel electrophoresis from 38 patients with AKI. Patients were randomly assigned to a training set, an internal test set, or an external validation set. Spot abundances were analyzed by artificial neural networks to identify biomarkers that differentiate between ATN and PRA. When the trained neural network algorithm was tested against the training data, it identified the diagnosis for 16 of 18 patients in the training set and all 10 patients in the internal test set. The accuracy was validated in the novel external set of patients where conditions of 9 of 10 patients were correctly diagnosed including 5 of 5 with ATN and 4 of 5 with PRA. Plasma retinol-binding protein was identified in 1 spot and a fragment of albumin and plasma retinol-binding protein in the other. These proteins are candidate markers for diagnostic assays of AKI.