Exposure to circulating cell-free hemoglobin is a ubiquitous feature of open-heart surgery on cardiopulmonary bypass circulation. This study aims to determine the origins and dynamics of circulating cell-free hemoglobin and its major scavenger proteins haptoglobin and hemopexin during neonatal cardiopulmonary bypass. Forty neonates with an isolated critical congenital heart defect were included in a single-center prospective observational study. Blood samples were obtained preoperatively, hourly during bypass circulation, after bypass separation, at admission to the pediatric intensive care unit, and at postoperative days 1–3. Concentrations of cell-free hemoglobin, haptoglobin and hemopexin were determined using ELISA. Neonates were exposed to significantly elevated plasma concentrations of cell-free hemoglobin and a concomitant depletion of scavenger protein supplies during open-heart surgery. The main predictor of cell-free hemoglobin exposure was the concentration of cell-free hemoglobin in blood prime solution. Concentrations of haptoglobin and hemopexin in prime solution were important determinants for intra- and postoperative circulating scavenger protein resources.