Background: Detection of cancer with improved discrimination compared to current blood tests could be achieved using an approach that assesses extracellular vesicles (EVs). This approach should have high sensitivity (se) because of EVs abundance in blood and high specificity (sp) by assaying EVs with multiple cancer-related protein and glycosylation epitopes (PGEs) co-localized on their surfaces. We are developing a platform technology that detects multiple cancer-related PGEs co-localized on the same EV using immunoaffinity-capture and proximity-ligation qPCR. In this study, we compare the performance of this technology vs plasma CA125 for correctly categorizing early-stage high-grade serous ovarian cancer (HGSOC) vs healthy/benign ovarian tumors (OT). Methods: We evaluated our EV-based platform technology using 7 PGE combinations to discriminate HGSOC from benign adnexal masses. We first derived a prediction model on a retrospectively collected cohort of 42 HGSOC and 26 benign OT samples from 2 commercial vendors and 24 healthy controls (HC) using a machine-learning algorithm. We validated this model on an independent cohort [89 HGSOC: Stage I (17), II (35), III (37); 192 benign OT] from university-associated biobanks and 124 HC. We also assessed the assay’s performance in plasma from 87 women with off-target cancers and 42 women with inflammatory conditions from commercial vendors. For each sample, we also measured CA125 levels using a commercial ELISA. Results: The prediction model distinguishes HGSOC from benign and HC with an AUC of 0.965 (95% CI 0.93-0.99), with 89.9% (0.82-0.95) se at 98% sp. For stage I/II HGSOC, the model achieves an AUC of 0.942 (0.9-0.99), with 84.6% (0.72-0.93) se at 98% sp. By comparison, CA125 achieves an AUC of 0.875 (0.81-0.94) and 44.2% (0.3-0.59) se at 98% sp. Direct comparison of CA125 and our model shows a significant difference at 98% sp for both all and stage I/II HGSOC (McNemar p-val < 0.001). When comparing HGSOC to HC, there is no significant difference between our model and CA125 (p-val = 1.0). There is a significant difference when comparing patients with all stage and stage I/II HGSOC to patients with benign OT (p-val < 0.001). Our assay had 1 false positive and CA125 had 3 false positives out of 42 inflammatory cases. Conclusions: These preliminary data suggest our platform technology for detecting PGEs co-localized on individual EVs may detect all stages of HGSOC from plasma with high se at a very high sp. Our assay may improve on CA125 by distinguishing stage I/II HGSOC from benign OT and could have clinical utility for both early detection and surgical referral recommendation for benign and malignant OT. While the diverse cohorts in this study may present challenges in interpretation, the reproducibility in an independent cohort is encouraging and supports further investigation using cases and controls from well-defined cohort studies.