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Objective: NIPT for fetal aneuploidy by digital PCR has been hampered by the large number of PCR reactions needed to meet statistical requirements, preventing clinical application. Here, we designed an octoplex droplet digital PCR (ddPCR) assay which allows increasing the number of available targets and thus overcomes statistical obstacles. Method: After technical optimization of the multiplex PCR on mixtures of trisomic and euploid DNA, we performed a validation study on samples of plasma DNA from 213 pregnant women. Molecular counting of circulating cell-free DNA was performed using a mix of hydrolysis probes targeting chromosome 21 and a reference chromosome. Results: The results of our validation experiments showed that ddPCR detected trisomy 21 even when the sample’s trisomic DNA content is as low as 5%. In a validation study of plasma samples from 213 pregnant women, ddPCR discriminated clearly between the trisomy 21 and the euploidy groups. Conclusion: Our results demonstrate that digital PCR can meet the requirements for non-invasive prenatal testing of trisomy 21. This approach is technically simple, relatively cheap, easy to implement in a diagnostic setting and compatible with ethical concerns regarding access to nucleotide sequence information. These advantages make it a potential technique of choice for population-wide screening for trisomy 21 in pregnant women. [ABSTRACT FROM AUTHOR]