Urinary interleukin-18 and cystatin-C are suggested to be biomarkers for predicting acute kidney injury (AKI). The aims of this study are to examine whether the urinary concentrations of interleukin-18 and cystatin-C vary with gestational age and other factors in non-AKI control neonates, and to determine whether urinary interleukin-18 and cystatin-C can predict AKI development in non-septic critically ill neonates, independently of potential confounders.We enrolled 62 non-septic critically ill neonates. Urine was collected every 48-72 h during the first 10 days of life.Urinary concentration of cystatin-C, but not interleukin-18, decreased with increasing gestational age and body weight, but not with increasing postnatal age in non-AKI control neonates. Both urinary interleukin-18 and cystatin-C were associated with AKI, even after controlling for gestational and postnatal age, birth weight, gender, Apgar score and the score for neonatal acute physiology in non-septic critically ill neonates. Urinary interleukin-18 and cystatin-C had odds ratios of 2.27 and 2.07, and achieved the area under-the-receiver-operating-characteristic curve of 0.72 and 0.92, respectively, for predicting AKI.The urinary concentration of cystatin-C, but not interleukin-18, may decrease with increasing renal maturity. Both urinary interleukin-18 and cystatin-C are independently predictive of AKI in non-septic critically ill neonates.