Background: Screening for OC that relies on CA125 and imaging lacks early-stage sensitivity, highlighting the need for a more effective test. EVs in blood may provide increased sensitivity from vesicles released in abundance and increased specificity from assessing multiple cancer-related markers colocalized on a vesicle surface. We report performance of an EV based OC Test in samples from asymptomatic postmenopausal women in the United Kingdom Collaborative Trial of Ovarian Cancer Screening (UKCTOCS). Methods: The OC Test (biomarkers, classifier, cutoff) was locked following a training study, after which we observed consistent assay performance in an independent cohort. Here we evaluated the OC Test in a blinded case-control study nested within the no screening (NS) and annual ultrasound screening (USS) arms of UKCTOCS. Cases were all women with a diagnosis (Dx) of high-grade serous carcinoma (HGSC) within 36 months following sample donation and were matched 10:1 by age at collection to women who had no OC detected before or within the trial follow up period (controls). In addition, samples drawn within 6 months prior to a false positive (FP) transvaginal ultrasound from women who underwent surgery for benign findings in the USS arm were evaluated. OC Test and research use CA125 test at previously established 98% specificity cutoff, were determined blinded to case-control status, with unblinding done by UCL only after all tests were completed. Results: We assessed 129 samples from women with HGSC, 1310 controls and 100 USS FP samples. Data from 160 samples were excluded due to operator error, low sample volume or quality issues. In healthy controls, we observed a 97.7% specificity for the OC Test, and 95.5% for CA125, P = 0.006 McNemar test. We observed a sensitivity of 82% for the OC Test in samples drawn 0-12 months prior to Dx, and 63% for CA125, P = 0.016 McNemar test. Additionally, only 2.3% (2/86, 95% CI 0.3-8.1) women who underwent surgery for false positive USS results were positive for the OC Test vs 10.4% (9/86, 95% CI 4.9-18.9) for CA125. Conclusions: The OC Test is capable of highly sensitive and specific detection of HGSC in asymptomatic postmenopausal women one year prior to Dx and can detect HGSC up to three years prior to Dx with superior sensitivity and specificity compared to CA125. The data supports further evaluation of the OC Test for OC screening.