The three major causes of anemia in neonates are blood loss, decreased red blood cell production, and increased degradation of erythrocytes. Establishing the cause of anemia in a neonate born prematurely can be challenging. Clinically, fetomaternal hemorrhage (FMH) can be difficult to diagnose-the condition often presents only after the manifestation of severe fetal anemia. FMH can be confirmed by determining the fetal hemoglobin F fraction in the mother, which is traditionally performed using the Kleihauer-Betke test (KBT). Herein, we present a case study of a newborn baby boy of Dutch ethnicity with massive FMH and negative KBT result. The KBT result appeared to be false-negative due to AO antagonism. However, the results of an additional marker alpha-fetoprotein (AFP) test confirmed the diagnosis of massive FMH. Therefore, measuring AFP in maternal blood can be helpful in confirming FMH in unexplained anemia of the neonate.