The study investigated an early non-invasive epigenetic approach for trisomy 21 risk assessment in maternal plasma using methylated cell-free DNA (mcf-DNA) and methylated extracellular DNA from vesicles (mev-DNA), targeting the fetal-specific methylated regions of RASSF1A, ERG and UMODL1 (U1 and U2). Blood samples were taken from 120 pregnant women at higher risk of trisomy 21 between 10 and 24 weeks of pregnancy, and karyotyping confirmed trisomy 21 in 3 of them. In addition, they analyzed mcf-DNA and mev-DNA from 8 healthy pregnant women, 8 non-pregnant women and age-matched women with trisomy. Fetal-specific methylated regions were assessed by qPCR method to compare cycle threshold (Ct) values and gene copy number analysis using plasmid standards. Trisomy 21 pregnancies had significantly lower mean Ct values for RASSF1A and UMODL1 (U1 and U2) in both mcf-DNA and mev-DNA compared to healthy pregnancies. The number of gene copies was higher in healthy pregnancies than in trisomic pregnancies and significantly higher in mcf-DNA than in mev-DNA for all tested genes. The results confirm the applicability of methylated DNA immunoprecipitation (MeDIP)-based qPCR assays in a North Indian population for non-invasive prenatal screening.