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Introduction: DNA methylation is the essential epigenetic mechanism of cell regulation, and one of the earliest signatures in cancer development. Blood-based DNA methylation has been tested for cancer early detection. Several methods have been used for DNA methylation sequencing, which include whole genome bisulfite sequencing (WGBS) and antibody-dependent DNA immunoprecipitation (MeDIP). However, WGBS has known issues of DNA loss and fragmentations, and MeDIP only captures the methylated DNA regions and are biased. Therefore, methods that can accurately quantify DNA methylation status while keeping DNA less damaged and loss are highly needed, especially for liquid biopsy at early cancer stages (due to its low cfDNA amount). Methods: We developed a probe capture-based cfDNA methylation assay using proprietary in-house developed liquid biopsy NGS platform and DeepSEA software algorithm. Analytic assay validation has been conducted using reference materials with known methylation profiles. Both cell line gDNA with known methylation profiles and plasma cfDNA from patients were used for this methylation study. Methylation profiles, DNA damages, and losses were evaluated and compared. Fragment distribution and copy number variations (CNV) were compared between untreated and treated samples. Results: In this study, we developed a cfDNA-based methylation assay that measures DNA methylation status and genomic profiling simultaneously. Using both cell line gDNA and plasma cfDNA, the assay detects both methylation change and genomic alterations, including fragment size distribution and CNV in a single test. Discussion: The successful development of cfDNA methylation assay could help identify cancer at its early stage. It also enables the detection and monitoring of minimal residual disease (MRD) and in obtaining information on therapeutic targets and resistance mechanisms relevant to the management of personalized cancer care. Citation Format: Binggang Xiang, Zhixin Zhao, Maya Stevens, Chao Dai, Feng Xie, Xiaohong Wang, Kemin Zhou, Shidong Jia, Pan Du, Jianjun Yu. Development of cfDNA-based methylation assay for early cancer detections [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3670.