Abstract Colorectal cancer (CRC) is the second most common cause of cancer deaths when men and women are combined in the U.S. Early detection in the precancerous stage is key to reducing the CRC morbidity and mortality rates. Thus, there is a critical need for a cost-effective, time-efficient, and convenient clinical tool for early CRC detection. We have developed a multiplex qPCR assay (ColoScapeTM) to detect CRC-associated genetic and epigenetic changes from liquid biopsy samples (i.e., cell-free DNA) using our proprietary QClamp® XNA technology. The QClamp® XNA technology is very unique and enables the ColoScapeTM assay to selectively amplify the mutant and methylated DNA target sequences by using a synthetic DNA analog XNA (Xenonucleic Acid). This study demonstrates the analytical performance for detecting low-abundant mutated and methylated gene copies, as well as assess the clinical performance using plasma cell-free DNA samples from patients with CRC or advanced adenomas (≥1 cm) and from individuals with normal colonoscopies. The ColoscapeTM test is currently designed to detect mutations in 8 genes with 61 mutations and 7 methylated markers. The test consists of two parts: (i) the detection of mutations in 8 genes (APC, KRAS, BRAF, TP53, CTNNB1, NRAS, SMAD4, and PIK3CA) and (ii) the detection of 7 methylation targeted genes. For the assay analytical performance, the ColoScapeTM test shows that XNA effectively suppressed the wild-type background amplifications and led to amplify mutation and methylation target sequences dominantly, providing a high level of analytical sensitivity and specificity. The ColoScapeTM test is highly reproducible with intra- and inter-assay coefficient of variation of <10% and the cross-reactivity within the assay was limited and negligible. The results for the assay analytical sensitivity indicated that each target could be detected between 0.1% and 0.5% variant allele frequency in 10 ng cfDNA. The preliminary assay clinical specificity and sensitivity were 100% (95% CI: 91.3-100%) and 86% (95% CI: 66-95%) respectively for CRC and 91% specificity (95% CI: 75%-98%) and 60% sensitivity (95% CI: 17%-93%) for advanced adenomas. In summary, the ColoScape test utilizing the XNA-based technology provides high sensitivity and high specificity to CRC and advanced adenomas with a great potential to be used as an early screening test. Citation Format: Hiromi Tanaka, Shuo Shen, Mauro Scimia, Larry Pastor, Jonathan Li, Andrew Y. Fu, Daniel Kim, Rui Ni, Aiguo Zhang, Michael Y. Sha. ColoScape test: a molecular assay to detect early-stage colorectal cancer in plasma cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6505.