Background/Objectives: Hemolysis has been associated with acute kidney injury (AKI) in infants and neonates after surgery involving cardiopulmonary bypass (CPB). Erythrocyte hemolysis and subsequent end-organ injury have been shown to be a complex process involving the liberation of multiple molecules that mediate the loss of nitric oxide and oxidative damage. This study assesses the association of multiple products of erythrocyte hemolysis with the evolution of AKI in neonates and infants undergoing CPB surgery. Methods: Blood and urine samples were collected at multiple time points before and after CPB and stored within an institutional biorepository. Twenty-one patients with AKI were matched with twenty-one non-Aki patients based on demographic and case complexity data. Results: Samples were analyzed for cell-free hemoglobin, heme, non-transferrin-bound iron, haptoglobin, hemopexin, and nitrite/nitrate. NGAL and KIM-1 were measured to index AKI. Cell-free hemoglobin was higher, haptoglobin was lower, and haptoglobin:hemoglobin ratio was lower in AKI compared to non-AKI patients. Conclusions: AKI in neonates and infants after CPB is associated with a pre and postoperative decrease in serum haptoglobin. These results confirm the need for future studies to prevent injury from hemolysis during CPB and potentially identify at-risk patients with decreased haptoglobin levels before surgery if delay is an option.