We conducted a prospective study in pediatric patients presenting to an emergency center (EC) to (1) test the ability of urinary acute kidney injury (AKI) biomarkers to predict AKI presence and severity and (2) determine if these biomarkers offer similar precision in patients with versus without a known baseline SCr. The accuracy of five putative urinary biomarkers to detect AKI presence and severity was evaluated in 252 children presenting to our EC. AKI was defined by the modified pediatric RIFLE (pRIFLE) system. Eighteen children had AKI by pRIFLE, yet 33–50% of these AKI cases may have been missed since the EC SCr was/dl. Urinary NGAL, Kidney Injury Molecule-1 (KIM-1) and beta-2 microglobulin (β2M) all demonstrated good to very good accuracy (AUC > 0.70 to 0.80) to predict patients with pRIFLE-Injury (>50% decrease in eCCl) versus patients with pRIFLE-Risk (25–50% decrease in eCCl) or without AKI. Our data suggest urinary biomarkers may serve well to detect AKI accurately in the pediatric EC setting, even in cases where SCr levels are normal. Further study is required to determine if these biomarkers obtained in the EC can predict AKI development or progression in hospitalized patients.