A universal method confirming the presence of circulating cell-free fetal (ccff) DNA in maternal plasma is important in the field of noninvasive prenatal diagnostics. Restriction endonuclease digestion of one allele of a single-nucleotide polymorphism (SNP) was used to allow detection of paternal alleles in maternal plasma DNA. Multiplexed genotyping of 92 panethnic high-frequency SNPs predicted >0.99 probability of detecting at least four informative loci per sample. Child-maternal paired DNA samples were used to confirm detection of 2% child's heterozygous DNA in a background of maternal DNA homozygous for the digestible allele. By restriction endonuclease digestion of DNA in a PCR cocktail before thermal cycling, 10 genomic copies of a paternal SNP allele were detectable in a background of 990 maternal SNP alleles. A comparison of 154 pregnant and nonpregnant female plasma DNA samples demonstrated enhanced detection of nondigestible SNP alleles in maternal plasma. Receiver operating characteristic curve analysis showed an optimal detection threshold of four nondigestible SNP alleles in plasma for the confirmation of ccff DNA and 98% sensitivity and 96% specificity at a 95% confidence level. Our study demonstrates the ability of this technique to confirm the presence of paternal alleles from ccff DNA in maternal plasma.