This study evaluated the effect of hyperbilirubinemia on hearing thresholds and the auditory pathway in preterm infants using serial auditory brainstem response (ABR) tests. Seventy preterm infants admitted to the neonatal intensive care unit for hyperbilirubinemia treatment between 2013-2017 were included. Serial ABR tests found higher unconjugated bilirubin concentrations were associated with abnormal ABR thresholds, indicating hearing loss. Unconjugated bilirubin levels also directly correlated with hearing thresholds on ABR. This study demonstrates that unconjugated bilirubin concentration is a better predictor of bilirubin-induced hearing loss in preterm infants than total serum bilirubin levels.