In very low birth weight (VLBW) infants, acute renal impairment (ARI) is common but there is no consensus about criteria for its diagnosis. Neutrophil gelatinase-associated lipocalin (NGAL) is an early and sensitive indicator of renal impairment in experimental animals, children, and adults. Urinary NGAL (UNGAL) is detectable in VLBW infants; however, there is no reference range in this population. The objective of this study is to define the reference range for UNGAL in VLBW infants with no risk factors for ARI. UNGAL concentration was determined in urine samples collected from day of life (DOL) 4 through DOL 30 in 50 newborns with uncomplicated clinical courses, selected from a total of 145 prospectively enrolled appropriate for gestational age (AGA) inborn VLBW premature infants. The birth weight and gestational age ranges were 790–1490 grams and 26–33 weeks, respectively. The median, 95th and 99th percentiles, and range of pooled UNGAL values were 5 ng/ml, 50 ng/ml, 120ng/ml, and 2–150 ng/ml, respectively. Greater variability and higher quantile levels of UNGAL were observed in females vs. males. In conclusion, a reference range for UNGAL in VLBW infants, similar to that in children and adults, has been established.