Abstract In the United States, lung cancer is the leading cause of cancer-related deaths. In recent decades, the advent of molecularly-targeted therapies in the management of non-small-cell lung cancer (NSCLC) has led to significant improvements in patient outcomes. As such, optimum care depends on timely molecular testing. The mutational spectrum associated with NSCLC, however, is complex and riddled with various point mutations, amplifications, and rearrangements/fusions across multiple genes. Comprehensive testing of NSCLC has historically required multiple testing modalities to detect all clinically-relevant molecular targets. At our institution, a combination of immunohistochemistry, fluorescence in situ hybridization (FISH), and sequencing-based tests are performed on all NSCLCs. Though fragmented, this approach ensures testing for all current targetable genetic alterations is performed. Unfortunately, this style of testing is often incompatible with the limited amount of tumor tissue acquired by biopsy. In 2021, our institution implemented PGDx elio tissue complete (PGDx), introducing redundancy in ROS1 translocation testing, previously assessed by FISH through send-out testing to Mayo Clinic. The objective of this study was to perform a retrospective analysis of our molecular testing experience for NSCLC, with a spotlight on the nuances of ROS1 testing. Since its implementation at our institution, PGDx has been performed on 172 cases of NSCLC. As part of our NSCLC reflex testing agreement, ROS1 translocation FISH testing was also attempted on all cases. Of the 172 cases, one clinically actionable ROS1 translocation was identified by PGDx (0.5%). Interestingly, FISH testing for this case was equivocal. The remaining cases were negative for ROS1 translocations. Notably, FISH failed in six cases (3.5%) due to insufficient tissue. Three additional cases (1.7%) had minimal tumor tissue available for FISH testing (less than 300 cells), providing suboptimal results. Altogether, PGDx was successfully performed on all samples and, in the 166 cases where FISH was successful, there was no discordance between orthogonal tests. This supports the validity and reliability of PGDx in identifying clinically relevant rearrangements/fusions in ROS1. In this study, we demonstrate that PGDx can effectively replace FISH testing for ROS1 translocations in NSCLC. In addition to being technically equivalent to FISH for ROS1 with comparable turnaround times, PGDx adds substantial benefits. Replacing FISH testing with PGDx improves the diagnostic workflow, improves tissue utilization, and eliminates the costs associated with send-out orders. The cost of Mayo ROS1 FISH testing averages $520 per sample and this amount is billed to patient insurance, though reimbursements vary. Further, PGDx can detect the ROS1 gene fusion partners, a feature not possible through routine FISH testing in NSCLC. This added clinical information reduces the ambiguity of equivocal FISH results, exemplified by our study. By implementing this change, our institution will optimize both diagnostic practices and resource utilization in the clinical setting.