There is evidence that size distribution of cell-free DNA (cfDNA) fragments can be diagnostically relevant. The present work describes a multiplex quantitative real-time polymerase chain reaction technique modified and validated by the authors to study the degree of cfDNA fragmentation in blood plasma. Based on the detection of Alu and hLINE-1 repeats, this technique employs fluorescent probes. We selected suitable primers and probes, optimized PCR conditions and estimated the dynamic range and sensitivity threshold of the assay. The modified PCR had a dynamic range of 6 logs, its efficiency being over 90 %. We demonstrated that cfDNA fragmentation index did not differ significantly between healthy women (n = 16) and women with stage III–IV ovarian cancer (n = 14). Therefore, further research on a larger sample is needed using electrophoretic cfDNA fractionation.