Abstract Papillary thyroid carcinoma (PTC) accounts for up to 90% of thyroid cancer cases, and its differentiation from benign conditions presents a challenge in clinical practice. Fine needle aspiration biopsy (FNAB) has become a recommended practice for evaluating thyroid nodules (TN) with imaging features suspicious of malignancy. Although FNAB demonstrates high accuracy in discriminating TN, a significant number are classified as malignant indeterminate (MTI), which can only be fully diagnosed by post-surgical histologic evaluation. Up to 30% of FNAB findings are expected to be MTI, leading to a high rate of unnecessary hemithyroidectomies in about 75% of patients. In PTC, the most common mutation is c.T1799A in the BRAF gene (rs113488022), resulting in V600E in the B-raf protein, leading to an increase expression by altering the MAPK pathway. The aim of this study was to evaluate the efficacy of discriminating patients with malignant and benign TNs by detecting BRAF V600E in tissue from FNAB for molecular diagnosis of PTC. The qPCR technique with allele-specific probes was used to detect the BRAF c.1799T>A mutation in somatic DNA samples extracted by FNAB from TN (n=99) with suspected malignancy by ultrasonography. Cytologic diagnosis of biopsies was performed using the Bethesda System (TBSRTC). Diagnostic accuracy measures for evaluation of BRAF c.1799T>A as a diagnostic tool were performed with MedCalc software. Of the 99 patients, it was determined by cytology that 50 patients (50.51%) had PTC and 49 benign neoplasms (49.49%). The mutation was detected in 28 of the PTC samples (56%) (AA homozygotes [n=12, 24%] and AT heterozygotes [n=16, 32%). No mutation carriers were found among patients with benign neoplasms. Regarding accuracy measures were 56% for sensitivity of 56% and 100% specificity. The positive predictive value was 100% and the negative predictive value was 69.01%, with an accuracy of 77.78%. Finally, the negative likelihood ratio (LR-) value was 0.44 (95% CI: 0.32 to 0.60), while the positive likelihood ratio (LR+) value could not be calculated as there were no patients with the mutation in the group of patients with benign TN. In conclusion, our results suggest that the presence of BRAF c.1799T>A mutation could be indicative of PTC, confirming its usefulness in the differentiation between malignant and benign tumors. BRAF V600E detection may be valuable as an adjunct to conventional cytology, improving diagnostic accuracy and reducing the need for unnecessary surgical procedures. While highlighting the potential utility of this technique as a diagnostic tool, further analysis and consideration of other metrics is recommended for a full evaluation of its clinical relevance. Citation Format: Anette R. Gastelum-Quiroz, Andrea Ross-Orozco, Marco A. Álvarez-Arrazola, Eliakym Arambula-Meraz, Karla L. Morales-Hernández, Hanna M. Tolosa-Lerma, Fred Luque-Ortega, Paola Y. Sánchez-Aboytes, Noemí García-Magallanes. qPCR in the diagnosis of papillary thyroid cancer: Assessment of BRAF V600E mutation in FNA biopsies of thyroid nodules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB276.