Trisomy 21 and trisomy 18 are the two most common chromosomal anomalies in live births. To find new biomarkers for aneuploidies and pathogenesis of fetal malformations, we measured insulin-like growth factor (IGF) axis-related proteins in amniotic fluid (AF) of pregnant women carrying trisomies 21 or 18 affected fetuses using multiple reaction monitoring (MRM) approach. Eighty-five AF samples from pregnant women carrying either trisomy 21, trisomy 18, or normal fetuses were collected. IGF axis-related proteins in AF after serial treatments were quantitated with MRM method. The differential protein levels were also confirmed by western blot in AF without any treatment. The IGF type I receptor and pregnancy-associated plasma protein-A in AF of trisomy 21 (1.35 ± 0.32 and 13.36 ± 3.64 µg/mg protein) and trisomy 18 (1.39 ± 0.40 and 12.80 ± 1.84 µg/mg protein) were decreased versus normal controls (2.16 ± 0.59 and 23.77 ± 6.18 µg/mg protein). IGF binding protein 5 was reduced in trisomy 18 (1.47 ± 0.33 vs 2.36 ± 0.77 µg/mg protein). These alterations were confirmed by western blot. The other proteins showed no significant difference between the three groups. Our data suggested that MRM can provide a powerful platform for the identification of biomarkers in AF that have crucial developmental effects in the aneuploid fetus.