Abstract Non-Small Cell Lung Cancer (NSCLC) is the most common type of lung cancer with 236,740 new cases diagnosed in 2022 in the United States. In this study, we demonstrate the performance of a digital PCR (dPCR) based assay that reports 15 (NCCN) biomarkers using only 1 slide of FFPE tissue. ChromaCode’s High Definition PCR (HDPCR™) NSCLC Tumor Profiling Panel (Research Use Only) detects guideline biomarkers including variants for EGFR, BRAF, KRAS, ERBB2, ALK, ROS1, RET, MET, and NTRK1-3 with a 24 hour turnaround time and a simple PCR workflow. A total of 34 NSCLC FFPE specimens were evaluated using the HDPCR NSCLC Panel. DNA and RNA were extracted from a single 10 µm section of specimen, with a sister section previously characterized with the Oncomine Precision Assay GX. The DNA/RNA eluates (20ng DNA per well, 20ng RNA) were run with the HDPCR NSCLC Panel on the Qiagen QIAcuity dPCR instrument. Data analysis was conducted using ChromaCloud™, which includes a proprietary algorithm that can detect and quantify 6 targets (5 targets and 1 internal control) for each of the 3 wells. DNA samples discordant with the comparator assay were evaluated using the Archer DNA VariantPlex panel, while RNA discordant samples were analyzed using the Archer RNA fusion plex panel. Of the 34 specimens evaluated, 16 SNV, 13 Insertion/Deletion, and 7 fusion positive specimens were identified using NGS. On the dPCR assay, the results show a high-level of agreement, after discordant resolution, of 95.8% across the panel. The minor allele fraction (MAF), also calculated using ChromaCode Cloud, ranged from 5% to 90%. With 20 ng input (Qubit), the average copy number of amplifiable DNA ranged from 791 copies (~2.3 ng) to 6583 copies (~19.9 ng) with a mean of 2410 copies (~7.43 ng). Here we demonstrate that the HDPCR NSCLC Tumor Profiling Panel provides comparable performance to validated NGS assays with a turnaround that enables adherence to guideline-recommended treatment, and a low sample requirement that maximizes the number of patients for molecular testing. The standard protocols used by the assay and the use of a cloud based analysis pipeline enables easy deployment of this assay in any lab. Citation Format: Kerri Cabrera, Jeff Gole, Bryan Leatham, Isabel Regoli, Tiffany Martinez, Andrew Richards, Leah Herdt, Juan Enciso, Paige Berroteran, Heather Carolan, Brad Brown. Performance of a 24-hour turnaround, guideline-complete NSCLC assay, optimized for sparse samples [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 2194.