The aim of this study was to identify differentially methylated regions in cell-free DNA (cfDNA) between healthy persons and patients with advanced stage ovarian cancer (ASOC) and to identify differences in cfDNA methylation before and after cytoreductive surgery. Plasma-derived cfDNA was analyzed by a high-throughput genome wide DNA methylation sequencing technique: MeD-seq. A training set of therapy naïve cfDNA samples of patients with ASOC (≥FIGO stage IIIB, n=10) was compared with cfDNA of healthy controls (n=10) to define a ASOC specific cfDNA methylation signature. A cumulative hypermethylation score was constructed and a validation set of pre- and postoperative samples of 39 patients were compared using this score. MeD-seq results of tumor tissue samples were correlated with cfDNA results. Patients with ASOC showed a clear distinct cfDNA methylation signature from healthy controls (p