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Minimally invasive next-generation sequencing-based (NGS) liquid biopsy (LB) tests using cell-free DNA (cfDNA) are increasingly becoming an important tool for the clinical management of cancer patients. The ability to accurately detect molecular alterations from a single plasma sample is valuable for biomarker discovery, diagnostics, and disease monitoring. However, challenges for this assay include low concentrations of circulating tumor DNA (ctDNA) within the cfDNA plasma fraction and PCR and sequencing errors, which can lead to decreased sensitivity in variant calling. Further, somatic mosaicism due to clonal hematopoiesis of indeterminate potential (CHIP) in plasma makes accurate interpretation of LB results a challenge. Incorrect classification of false positives can lead to inappropriate therapeutic management. Here, we present an LB assay composed of a proprietary error reduction algorithm designed to eliminate false positives due to PCR and NGS errors and a white blood cell sequencing (WBC-seq) component to detect and bioinformatically filter out CHIP-related mutations. Consisting of a custom panel of 216 FDA/NCCN pan-cancer genes, our cfDNA LB assay was analytically validated with commercially available reference standards and human cell lines. At a clinically relevant sequencing depth of 4000x, we observed a limit of detection (LOD) of 0.2% variant allele frequency for single nucleotide variants (SNVs) and insertions and deletions (indels) with overall accuracies of 98% and 99% and sensitivities of 91% and 83%, respectively. To assess the LB assay performance on clinical samples, orthogonal whole-exome sequencing (WES) with subsequent standard somatic variant calling was performed on paired peripheral blood lymphocytes (PBLs) and plasma samples from 12 patients. The WES assay results were concordant with those obtained using our LB assay for SNVs (CCC = 0.75) and indels (CCC = 0.90). Further, our assay detected an additional 53% and 50% SNVs and indels, respectively, compared to WES. Finally, 8 out of 11 clinically actionable mutations were detected by both WES and the LB assay, while 3 additional SNVs were detected in the LB assay. Taken together, the use of WBC-seq to filter CHIP-derived mutations combined with our error reduction algorithm resulted in a substantial reduction in false positive variant calls without a compromise in the sensitivity or classification accuracy of SNVs and indels. Concordance was observed between traditional WES analysis and the LB assay developed here. These findings highlight the potential for our LB cfDNA assay to be used to improve personalized therapeutic options and guide clinical decision making. Citation Format: Anastasiya Yudina, Alexey Efremov, Danielle Sookiasian, Ekaterina Nuzhdina, Svetlana Podsvirova, Madison Chasse, Tori Conroy, Noel English, Sergey Starikov, Olesia Klimchuk, Dmitry Tabakov, Anna Love, Kushal Suryamohan, Artur Baisangurov, Cagdas Tazearslan, Nathan Fowler, Alexander Bagaev. Analytical validation of a liquid biopsy test using cell-free circulating tumor DNA for mutational profiling [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 1042.