Abstract Background: Circulating cell-free DNA (cfDNA) is present in low concentrations in healthy individuals. However, the molecular baseline of circulating variant allele fractions (cVAFs) in healthy individuals remains poorly defined. This study used a novel ultra-sensitive liquid biopsy platform to quantify cVAFs of common cancer driver mutations in a healthy population and compared them to profiles in patients with solid tumors. Methods: A total of 415 participants were enrolled, including 246 healthy individuals (59.3%) and 169 patients (40.7%) with solid tumors from seven primary sites: cervix, endometrium, ovary, colorectum, stomach, pancreas and prostate. Samples were prospectively collected at Korea University Anam Hospital (IRB No. 2022AN0090). Variant allele fractions were measured using MUTE-Seq, targeting key cancer-associated genes: TP53, IDH1, PIK3CA, KRAS, MYCN, NRAS, EGFR, BRAF, CTNNB, ERBB2, APC, AKT1, BRCA and PDGFRA. Comparisons were performed based on health status, cancer type, stage and involved organ system. Two metrics were used to quantify mutation burden: cVAF sum and cVAF mean, defined respectively as the total and average variant allele fraction of detectable mutations per individual. Results: Mutations were detected in 105 healthy individuals (42.7%) and 108 cancer patients (63.9%). In the healthy group, the average cVAF sum was 0.060 and the average cVAF mean was 0.041. In comparison, cancer patients showed substantially higher values, with an average cVAF sum of 1.380 and an average cVAF mean of 0.987. Median cVAF sum and mean values were also significantly higher in cancer patients compared to healthy individuals (0.000 to 0.076 for cVAF sum; 0.000 to 0.049 for cVAF mean; both p < 0.001). This difference in cVAF sum remained significant when the analysis was limited to participants with detectable driver mutations, with higher levels observed in cancer patients compared to healthy individuals (p < 0.001), whereas cVAF mean did not differ significantly between the groups (p = 0.537). A progressive increase in cVAF levels was observed across clinical groups, analysis showing that cVAF sum was higher in both early- and advanced-stage cancer patients compared to healthy individuals (p = 0.018 and p < 0.001, respectively). Subgroup analyses showed that colorectal, endometrial, ovarian, and pancreatic cancers had higher cVAF mean values than healthy participants. When grouped by organ system, both gynecologic and gastrointestinal tumors exhibited significantly elevated cVAFs compared to the healthy cohort (p < 0.001). Conclusions: This study defines a molecular reference range for cVAFs in healthy individuals. The presence of low-level mutations in nearly half of healthy individuals emphasizes the need for quantitative context in cfDNA-based diagnostics. These findings support the clinical utility of cVAF profiling and provide a benchmark for distinguishing pathological signals from background variation. Citation Format: Jungmin Kim, Sungjae An, In Seon Lee, Kyung-Hak Choi, Sunghyeok Ye, Junseok W. Hur. Comparative analysis of circulating variant allele fractions in healthy individuals and patients with solid tumors using an ultra-sensitive liquid biopsy assay MUTE-Seq [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A012.