Background/Aims: To evaluate serum prolactin and CA-125 levels as biomarkers for the diagnosis of peritoneal endometriosis. Methods: A prospective study was performed. Blood samples were drawn from a peripheral vein during the secretory phase of the menstrual cycle (day 19-21 prior to the surgery) to analyze through relative operating characteristic curve the serum prolactin and CA-125 levels for diagnosis of peritoneal endometriosis. The study wasperformed with 97 participants, 63 women with peritoneal endometriosis and 34 healthy women. Results: The sensitivity and specificity of peritoneal endometriosis diagnosis were equivalent for prolactin (21 and 99%) and for CA-125 (27 and 97%; p = 0.58). These two markers were used in a parallel test utilizing the usual cutoff (prolactin 20.0 ng/ml and CA-125 35 U/I). The sensitivity and specificity were 44 and 99%. However, by utilizing the best cutoff (prolactin 14.8 ng/ml and for CA-125 19.8 U/I), sensitivity, specificity and negative predictive value were 77, 88 and 97%, respectively. Conclusion: Serum CA-125 and prolactin levels assessed together, and considering the cutoff for CA-125 (19.9 U/I) and prolactin (14.8 ng/ml), allow the diagnosis of peritoneal endometriosis with acceptable sensitivity and specificity (77 and 88%) and a high negative predictive value (97%).