In infants and children, elevated whole blood zinc protoporphyrin/heme (ZnPP/H) measures iron-deficient (ID) erythropoiesis. Because immature erythrocytes are less dense than mature erythrocytes, we hypothesized that the sensitivity of ZnPP/H is improved if measured in the least dense cells. Blood was collected from control suckling, mildly and severely ID suckling rats. Cord blood was collected after uncomplicated pregnancies (control), diabetic pregnancies (severe ID) and after pregnancies at-risk for iron deficiency (mild ID). ZnPP/H was measured before and after a two-step density centrifugation to obtain the lightest 6.25% of erythrocyte (top fraction). The difference between whole blood and top fraction was defined as ΔZnPP/H. In rats, although the whole or top ZnPP/H differed by postnatal age, ΔZnPP/H was greatest after the interval with least body iron accrual. In either rats or humans with mild ID, whole blood ZnPP/H was similar to, but ΔZnPP/H was greater than controls. In rats and newborn humans, ΔZnPP/H is more sensitive than whole blood ZnPP/H in identifying conditions associated with impaired erythrocyte iron delivery and may become a useful tool in measuring erythrocyte iron incorporation in early development.