Abstract Monitoring measurable residual disease (MRD) via liquid biopsy is a promising method for catching early stage cancer and disease relapse long before traditional diagnostics, which generally require significant disease progression for detection. Assays in development include those that target patient-specific variants and fixed panels for all patients. Regardless, detection of variant allele frequencies at extremely low levels, well below the limit of detection of typical circulating tumor DNA (ctDNA) assays, presents a challenge that can be surmounted with well-designed reference materials that allow for assessment of sensitivity and specificity. We developed the Seraseq® ctDNA MRD Panel kit composed of 4 tumor fractions at decreasing levels to meet validation needs of both patient-specific and fixed panel targeted cfDNA NGS MRD assays. Genomic DNA from tumor and their SNP-matched normal cell lines, characterized by whole exome sequencing (WES), was fragmented to approximate the size of circulating cell free DNA (ccfDNA). This DNA was blended at tumor fractions (TF) of 0.5%, 0.05%, and 0.005% and biosynthetic DNA fragments containing more common and clinically relevant variants were spiked in at similar TF levels. A normal (0% tumor) ctDNA reference material was produced for comparison and identification of background variants. The VAFs for the biosynthetic variants were determined by digital PCR (BioRad QX200) and targeted cfDNA NGS assay (ArcherDx LiquidPlex) in the 0.5% TF mix. Somatic variants from the tumor DNA were assessed using a custom Agilent SureSelect XT HS2 assay targeting ~100 variants in the MRD panel mix using 50 ng as input. Variants were called at 1 observation. All biosynthetic variants were detectable in the TF 0.5% mix at anticipated VAFs (dPCR: 0.43% ± 0.12% and NGS: 0.33% ± 0.15%). Somatic VAFs of the ctDNA MRD panel mixes were measured at expected levels although not all variants were detected below 0.05% due to sampling. Agilent SureSelect XT HS2 libraries had ~25% incorporation efficiency and ~ 72% on-target. The 0.5% and 0.05% mixes showed evidence of more of the somatic variants at 1 or more copies than the other samples. The 0.005% mix showed evidence of more of the somatic variants than the 0% mix. The Seraseq ctDNA MRD Panel Mixes have been designed to provide a broad range of DNA mutations from tumor-normal matched cell lines and spike-in variants to aid sensitivity and specificity when applied to MRD evaluation of patient-specific and fixed panel cfDNA NGS MRD assays. Citation Format: Dana Ruminski Lowe, Benedicta Forson, Matthew G. Butler, Yves Konigshofer, Catherine Huang, Omoshile Clement, Russell K. Garlick, Bharathi Anekella. Reference materials for ctDNA-based measurable residual disease (MRD) assay development and validation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2219.