Abstract: The invention relates to a method for obtaining a set of reference samples and/or a set of reference parameters for the diagnosis of fetal aneuploidy from a maternal biological sample, containing cell-free DNA, said method comprising:—extracting cell-free DNA from a set of biological samples obtained from euploid pregnant women carrying a euploid fetus;—after the extraction step, analyzing the size distribution of the DNA molecules within each sample and selecting a set of samples based on the size distribution of the DNA molecules within said samples;—performing a massively parallel sequencing of DNA of each size-selected sample;—mapping the obtained sequences to the human genome for each sample;—calculating a set of reference parameters, wherein each reference parameter is indicative of the number of unique exact sequences mapped to a chromosome or chromosomal region of interest for each sample;—obtaining a set of reference samples and/or a set of reference parameters.