Gestational diabetes mellitus (GDM) is a state of hyperglycaemia and increased oxidative stress with onset during pregnancy. Human serum albumin (HSA) was extracted from 26 GDM and 26 nonGDM amniotic fluid samples collected at 15 weeks gestation and analyzed by mass spectrometry. The majority of all albumin isoforms were oxidized with the cysteinylated HSA as the base peak in the deconvoluted spectrum. The HSA peak areas, from a control sample, had 36% relative standard deviation (RSD) across the six experimental days but using the relative isoform distribution improved the precision to 3-6%. The results show that the relative contribution of permanently oxidized HSA was greater (P = 0.002) and reversibly oxidized HSA was lower (P = 0.006) for GDM compared to nonGDM in the samples measured. This implies that the path toward GDM has been set prior to 15 weeks gestation and results in increased protein oxidation.