Ovarian cancer is the fourth most common cancer in women worldwide. It is very heterogeneous at the clinical, histopathological and molecular levels and is caused by the accumulation of genetic and epigenetic changes in regulatory genes. More than 90% of ovarian cancers are epithelial in origin. Ovarian cancer is typically asymptomatic in its early stages, and, due to difficulties in early detection, most ovarian cancers are diagnosed at an advanced stage. The positive predictive value of CA-125, a routinely used serum protein marker, is < 30%; therefore, for effective screening, there is a need to develop a marker with high sensitivity for early detection. Development of blood-based biomarkers that detect DNA methylation in cell-free tumor-specific DNA is now being considered as a potential approach for the early diagnosis of cancer. Our objective in this study was to develop an absolute quantitative method, the MethyLight assay, to detect the promoter methylation status of two tumor suppressor genes. We analyzed the methylation level of the promoter regions of these genes in 42 tumor samples using the MethyLight assay. SOX1 promoter methylation was significantly higher in cancer samples than in normal samples (P = 0.011), whereas this difference between cancer and normal samples was not significant for DAPK1 promoter methylation (P = 0.18), when analyzed separately in a singleplex assay, whereas the detection frequency and significance level increased several-fold when these genes were analyzed together in a multiplex assay (P = 0.0004). The sensitivity was found to be 62% and 83% for DAPK1 and SOX1, respectively, when analyzed separately in the singleplex assay, but increased to 90% in the multiplex assay when either or both of the SOX1 and the DAPK1 gene promoters showed methylation.