The characterization of cord blood hemoglobin at the molecular level is a daunting challenge because hemoglobin F (HbF) and hemoglobin A (HbA) coexist in neonatal blood. We developed and validated a method using electrospray time-of-flight mass spectrometry (ES-TOF-MS) that measures, in a single analysis, relative levels of glycated and acetylated hemoglobin and allows the calculation of relative proportions of HbA, HbF(0), and HbF(1) in cord blood. Specific sections of acquired spectra were deconvoluted using a maximum entropy-based approach to true mass scale spectra. Mass precisions were less than 3 ppm with similar accuracies. Intra-interday precisions for α- and γ-chain glycation levels were 2.10%/3.72% and 2.75%/6.79%, respectively. The linearity of the α-chain glycation response was excellent (r(2) = 0.9990). We performed sample analysis on 39 cord blood specimens and found that the glycated α- and γ-chain levels were 2.27 ± 0.21% and 2.38 ± 0.29%, respectively, while the acetylated (G)γ and (A)γ-chain levels were 8.48 ± 0.53% and 7.14 ± 0.74%, respectively. We observed three types of HbF distinguishable by the intensities of γ-chain variants. Two-thirds of cord blood specimens were classified as HbF(I) with an intensity ratio (G)γ/(A)γ of 1.90 ± 0.12. For HbF(II) type (10/39 neonates), the intensity ratio of (G)γ/(A)γ was 3.71 ± 0.28. For three neonates with HbF(III), no (A)γ-chain was detected.