Haemophilia A is the most common X-linked recessive bleeding disorder. In 5% of severely affected patients the mutations responsible for the disease are large deletions encompassing from one exon to the complete Factor 8 (F8) gene. Large deletions in a male haemophilic patient are easily detected by the absence of the corresponding PCR product. However, in female carriers, identification of the various heterozygous large deletions is difficult representing a major limitation to accurate carrier diagnosis. The deletion is masked by the presence of the second allele that serves as template for the PCR reaction. Quantitative PCR can differentiate between the presence of one or two alleles. Here we report an assay based on multiplex amplification of several exons of the F8 gene of various length and subsequent quantitative evaluation of the amplicons by liquid chromatogphy (LC). Using this approach we achieved an accurate classification of 16 female carriers and eight non-carriers for deletions in the F8 gene in 19 investigated families. One mother and one grandmother were classified as non-carriers, underlining the high de novo mutation rate of large deletions in female germ cells. The large deletions in three families were confirmed by fluorescent in situ hybridization. In conclusion, the multiplex PCR-LC technique represents a rapid, simple and reliable method for detection of heterozygous large deletions in female carriers.