Objective: To develop a non-invasive diagnostic test for endometriosis. Design: We conducted two studies: the development study (study 1) aimed at (i) evaluating the ability of CA125, brain-derived neurotrophic factor (BDNF) and clinical variables in segregating between cases and controls and (ii) developing a diagnostic algorithm based on those results. In the validation study (study 2), the clinical performance of the developed in vitro diagnostic (IVD) test, in diagnosing endometriosis was validated. Interventions: Serum samples and clinical variables extracted from psychometric questionnaires were collected from the Oxford Endometriosis CaRe Centre biobank (UK). Case/control classification was performed based on laparoscopy and histological verification of endometrial glands and/or stroma in the excised lesions. Main outcome measures: Study 1 and 2 included n=204 and n=79 patients, respectively. CA125 and BDNF concentrations were determined using the endometriosis IVD ELISA kit. In study 2, serum CA125 and BDNF concentrations and clinical variables were introduced into the IVD test software hosting the data treatment algorithm, which generates the qualitative diagnostic result (positive or negative). Results: Results from study 1 showed that, for both biomarkers levels, a statistically significant difference was found between cases and controls. Among all clinical variables related to patients medical history considered, six were significantly different between cases and controls: record of a previous surgery to investigate endometriosis presence, painful periods leading to referral for endometriosis presence, severity of menstrual pain during last cycle, age at first experience of intercourse pain, age at first regular use of painkillers and age at first diagnosis of ovarian cyst. In study 2, the novel endometriosis IVD test demonstrated sensitivity and specificity values of 46.2% (25.5-66.8%) and 100% (86.7-100%), respectively. Conclusion: BDNF and CA125, together with patient clinical variables, allowed efficient segregation between controls and endometriosis cases. Due to its high specificity, the novel endometriosis IVD test is an accurate rule-in non-invasive method, potentially contributing to diagnose endometriosis.