Abstract Background: Detection of circulating tumor DNA (ctDNA) has demonstrated clinical utility as a prognostic marker for recurrence by indicating minimal residual disease (MRD). Because the abundance of ctDNA is below the detection limit (LoD) of current tumor genotyping methods (<0.1%), novel technology is required. Currently developed methods adopted personalized tumor genotype informed (tumor-informed) strategy with suboptimal LoD (∼0.01%). Increasing MRD positivity by lowering LoD is required to screen high-risk recurrence patients. Additionally, most of the currently developed MRD test lack clinically actionable information. Methods: A clinically validated personalized large-scale mutation profiling strategy MRD platform, CancerDetectTM, was improved by adding actionable mutation targets across 15 genes recurrently mutated in solid cancers. For analytical validation of personalized mutation regions, well-characterized mixtures of two germline gDNA (NA12891, NA12892) mixture were used, varying mixing ration from 0%, 0.0001%, 0.001%, 0.005%, 0.01%, 0.05% and 0.1%. For analytical validation of the clinically actionable regions, commercially available reference standard (Seraseq ctDNA MRD Panel Mix) was used with dilution ranged from 0.01% to 0.5%. The panel was designed to target more than 100 different genotype loci (Twist Biosciences). Results: The new iteration of CancerDetectTM integrates personalized tumor-informed large-scale mutation profiling strategy and drug target mutations (tumor-agnostic region) targeting strategy. Results showed that the limit of detection (LoD95) was reached down to 0.001% for tumor-informed region with 99.9% specificity and 0.1% for drug target mutations with 95% specificity. The precision analysis showed 100% and interference test showed that the test performance was not affected by interfering materials (Bilirubin. Wash Buffer) near LoD fraction. Increased sensitivity achieved MRD positivity if 27.2% in a prospective cohort of 551 patients with stage II/III CRC after one month of curative surgery and 45.2% in stage II/III gastric cancer after 3-10 weeks of surgery. Conclusion: The new iteration of CancerDetectTM showed the limit of detection of 0.001% with 99.9% specificity for tumor-informed region and 0.1% with 95% specificity for the tumor-agnostic region. The test result was not affected by the interfering materials and precision analysis showed 100% result. This indicates the new iteration of CancerDetectTM is a robust, sensitive MRD test. Citation Format: Sunghoon Heo, Seon-Kyu Ham, Hayoon Lee, Hwang-Phill Kim, Duhee Bang, Sang-Hyun Song, Tae-You Kim. Analytical validation of CancerDetect RUO assay: a minimal residual disease detection test that integrates tumor-informed and tumor-agnostic approaches with large-scale mutation monitoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3240.