Abstract Screening for ovarian cancer has been hindered by the lack of a clinical assay with sufficient sensitivity and specificity for use in the general population. Since bodily fluids in close proximity to tumors may contain higher concentrations of tumor biomarkers than blood samples, we hypothesized that proteins shed by ovarian cancer tumors could be detected in cervical effluent using the standard Pap test fixative and mass spectrometry (MS)-based proteomic techniques. Briefly, proteins were isolated from SurePathTM liquid-based Pap tests (Becton Dickinson) that were collected from 20 women with high grade serous ovarian cancer (HGSOC) and 10 women with benign ovarian conditions in the gynecologic oncology clinic, as well as 10 healthy women in the women’s clinic. Samples were labeled with tandem mass tags (TMT), fractionated with high pH reversed-phase liquid chromatography (LC), and then analyzed by LC-MS/MS analysis. The resulting spectra were searched against the UniProt human database to obtain quantitative measurements of >3000 proteins. By ANOVA, we found 30 proteins were differentially expressed in the ovarian cancer samples compared to both the benign and healthy samples (non-corrected p-value <0.05). Heavy peptide standards for these 30 proteins (5 candidate sequences per protein) were synthesized (Vivitide) to construct targeted MS assays using selected reaction monitoring (SRM). Peptides from 54 proteins that were previously identified in ovarian cancer tissues from a Clinical Proteomic Tumor Analysis Consortium study conducted at the Pacific Northwest National Laboratory were also included in the SRM assay. Multiplexed SRM analysis was then performed using a panel containing 244 peptides corresponding to 84 candidate biomarker proteins to quantify their abundance in 90 liquid-based Pap tests (40 HGSOC, 30 benign, and 20 healthy). Thirty-seven proteins showed a significant increase in the Pap test samples from women with HGSOC compared to healthy controls. A multi-protein classifier was then developed in which the combination of 6 proteins resulted in an AUC of 0.880 (95% CI: 0.738-0.989). The sensitivity at 90% specificity was 0.800 (95% CI: 0.470-1.00). In conclusion, in this proof of principle study we have demonstrated the translational potential of this approach, pending validation in prospectively collected samples. In the future, incorporating MS-based proteomics into routine Pap testing may allow women to be screened simultaneously for cervical and ovarian cancer. Citation Format: Amy P.N. Skubitz, Kristin L.M. Boylan, Ashley J. Petersen, Joshua R. Hansen, Tao Liu, Yuqian Gao, Yi-Ting Wang, Karin D. Rodland, Melissa A. Geller, Peter A. Argenta, Samantha L. Hoffman, Paul D. Piehowski. Identification of ovarian cancer protein biomarkers in liquid-based Pap tests [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B039.