The purposes of this study were to examine the accuracy of fetal hemoglobin (Hb F, alpha2gamma2) as quickly measured by a hemoximeter but verified by high performance liquid chromatography [HPLC, including Hb F total (Hb Ft), acetylated Hb F (Hb F1), and non acetylated Hb F ( Hb F*)], and to predict the Hb F levels for different gestational weeks of neonates. Thirty-nine neonates of predominantly Hispanic and African American ethnicity, with gestational ages ranging from 25 to 38 weeks, were investigated. Analyses were performed on 163 blood samples that were pure neonates' blood before the transfusion of any adult blood. Two neonates had increased Hb C [beta6(A3)Glu-->Lys, GAC-->AAG] levels (1.67-2.79%) and one neonate whose mother drank alcohol during pregnancy, had elevated Hb A2 levels (0.12-0.14%). After excluding these data points, the mean Hb F were overestimated by hemoximeter, 118.4 +/- 8.77% vs. 92.6 +/- 2.77% by HPLC (mean difference: 25.8 +/- 7.71%, p = <0.001). Mean Hb F1 was 10.5 +/- 2.28%. Hb F levels decreased as gestational age increased (p <0.001 for Hb Ft and Hb F*; p = <0.05 for Hb F1). A multivariate regression model for Hb F prediction was established with the best R2. The gestational age and post birth hours in the prediction of Hb Ft was included when Hb F could be determined at the clinical settings. Future studies may be needed to account for Hb F1 when measuring Hb F levels to assess oxygenation status in (pre term) neonates.