Abstract Background: A considerable number of estrogen receptor-positive breast cancer (ER+ BrCa) patients develop resistance to endocrine treatment. One of the most important resistance mechanisms is the presence of ESR1 mutations. The aim of the current study was the development and analytical validation of a highly sensitive and specific NaME-PrO-assisted ARMS (NAPA) assay for the detection of four ESR1 mutations (Y537S, Y537C, Y537N and D538G) in circulating tumor cells (CTCs) and corresponding plasma circulating tumor DNA (ctDNA) in patients with ER+ BrCa. Methods: We first developed the assay and validated the analytical specificity, analytical sensitivity and reproducibility. We further evaluated the performance of the developed assay in CTCs and ctDNA derived from 13 ER+ BrCa primary tumor tissues and 64 liquid biopsy samples: 32 EpCAM-isolated cell fractions and 32 corresponding plasma cell free DNA (cfDNA) obtained at different time points from 8 ER+ metastatic breast cancer patients, during a 5-year follow-up period and peripheral blood from 11 healthy donors (HD). We further compared the performance of the ESR1 NAPA assay with drop-off droplet digital PCR (ddPCR), using identical samples. Results: The developed assay is highly sensitive (detection of mutation-allelic-frequency of 0.5% for D538G and 0.1% for Y537S, Y537C, Y537N), and highly specific (0/13 mammoplasties and 0/11 HD for all mutations). In plasma ctDNA, ESR1 mutations were not identified at the baseline whereas the D538G mutation was detected in five sequential cfDNA samples during the follow-up period in the same patient. The developed assay gave comparable results with ddPCR, since the concordance between the ESR1 NAPA assay and drop-off ddPCR as evaluated using 32 identical cfDNA samples was 90.6% (29/32). In EpCAM-isolated cell fractions only the Y537C mutation was detected in one patient sample at baseline. Conclusions: We present a low cost, highly specific, sensitive and robust assay for blood-based ESR1 profiling. The developed assay is fast, with a comparable sensitivity to ddPCR but has lower cost relative to ddPCR, and thus can be used as a fast screening method to classify patients as positive or negative for ESR1 mutations. Our results are consistent with reports that indicate that ESR1 mutations (especially D538G, Y537S) are associated with more aggressive disease. Citation Format: Dimitra Stergiopoulou, Athina Markou, Eleni Tzanikou, Ioannis Ladas, G. Mike Makrigiorgos, Vassilis Georgoulias, Evi Lianidou. Development and analytical validation of a highly sensitive and specific NAPA assay for the detection of ESR1mutations in circulating tumor cells and plasma circulating tumor DNA [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 5160.