Pleural effusions (PE) are a common clinical problem. The discrimination between benign (BPE), malignant (MPE) and paramalignant (PPE) pleural effusions is highly important to ensure appropriate patient treatment. Today, cytology is the gold standard for diagnosing malignant pleural effusions. However, its sensitivity is limited due to the sometimes low abundance of tumor cells and the challenging assessment of cell morphology in cytological samples. This study aimed to develop and validate a diagnostic test, which allows for the highly specific detection of malignant cells in pleural effusions based on the DNA methylation biomarkers SHOX2 and SEPT9. A quantitative real-time PCR assay was developed which enabled the accurate and sensitive detection of SHOX2 and SEPT9 in PEs. Cytological and DNA methylation analyses were conducted in a case control study comprised of PEs from 114 patients (58 cases, 56 controls). Cytological analysis as well as SHOX2 and SEPT9 methylation resulted in 100% specificity. 21% of the cases were cytologically positive and 26% were SHOX2 or SEPT9 methylation positive. The combined analysis of cytology and DNA methylation resulted in an increase of 71% positively classified PEs from cancer patients as compared to cytological analysis alone. The absolute sensitivity of cytology and DNA methylation was not determinable due to the lack of an appropriate gold standard diagnostic for distinguishing between MPEs and PPEs. Therefore, it was unclear which PEs from cancer patients were malignant (containing tumor cells) and which PEs were paramalignant and resulted from benign conditions in cancer patients, respectively. Furthermore, DNA methylation analysis in PEs allowed the prognosis of the overall survival in cancer patients (Kaplan-Meier analysis, log rank test, p=0.02 (SHOX2), p=0.02 (SEPT9)). The developed test may be used as a diagnostic and prognostic adjunct to existing clinical and cytopathological investigations in patients with PEs of unclear etiology.