Abstract While there is no shortage of approved targeted therapeutics to address driver mutations in NSCLC, many patients are not tested for these alterations due to gaps in biomarker testing access. Challenges in NSCLC molecular testing include the need for assessment of multiple variants in both DNA and RNA, samples are often small specimens, cost, and clinical need for rapid turn-around time when patients are deteriorating and require timely treatment initiation. ASPYRE-Lung detects a targeted panel of 114 actionable genomic variants across 11 genes, covering NCCN treatment guideline-recommended biomarkers for samples that are associated with NSCLC. The assay simultaneously analyzes DNA and RNA with inputs of only 20 ng and 6 ng respectively. ASPYRE-Lung is easily adoptable by standard clinical laboratories, requiring only PCR and qPCR thermocyclers, with no specialist bioinformatics skills required for analysis and a run time of 2 days from specimen to result. We established the assay at two external sites and Biofidelity laboratories, with multiple operators at each site. We first determined that the assay had acceptable performance at each site, using a panel of contrived control samples. We then examined 56 matched DNA and RNA samples extracted from clinical FFPE lung tissue specimens. We also tested 29 unmatched samples derived from FFPE tissue (13), fine needle aspirate (FNA, 4), FNA rinse (5), peritoneal fluid (1), pleural effusion (1), pleural fluid (1) and fresh tissue (4), at all 3 sites. Data were analyzed using a simple cloud-based turnkey analysis solution. All samples yielded valid results across all runs. We assessed reproducibility (inter-run precision) using all 85 clinical samples, across new and experienced users, achieving a positive percent agreement (PPA) of 100% and negative percent agreement (NPA) of 99.99%. We then assessed concordance with NGS results, investigating any discrepancies. The 69 FFPE tissue-derived samples (56 matched DNA/RNA, 7 unmatched DNA, and 6 TNA) had PPA 96.55% and NPA 99.98%. The 16 cytopathology-derived samples had PPA 100% and NPA 99.96%. Together, these results demonstrate high concordance of ASPYRE-Lung and NGS across different types of clinical samples, alongside ease of adoption of the ASPYRE-Lung assay with simple training and no specialist expertise required. ASPYRE-Lung promises to enable all patients with NSCLC to start appropriate treatment in a cost-effective and timely manner. Citation Format: Sarah E. Herlihy, Caren Gentile, Samantha J. Scott, Brandon A. Smith, Kathryn A. Stoll, Kala F. Schilter, Justyna Mordaka, Rebecca Palmer, Christina Xyrafaki, Elizabeth Gillon-Zhang, Candace King, Ryan Evans, Amanda Green, Ana-Luisa Silva, Magdalena Stolarek-Januszkiewicz, Kristine von Bargen, Iyelola Turner, Chau Ha Ho, Alejandra Collazos, Nicola Potts, Dilyara Nugent, Jinsy Jose, Eleanor Gray, Elyse Shapiro, Wendy J. Levin, Aishling Cooke, Barnaby Balmforth, Robert Osborne, Honey V. Reddi, Vivianna M. Van Deerlin. Deployment of ASPYRE-Lung targeted variant panel across three sites and testing with FFPE tissue and cytology-derived nucleic acid samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5164.