The detection ofROS1rearrangements in metastatic non-squamous non-small cell lung carcinoma (NS-NSCLC) permits administration of efficient targeted therapy. Detection is based on a testing algorithm associated with ROS1 immunohistochemistry (IHC) screening followed byROS1FISH and/or next generation sequencing (NGS) to confirm positivity. However, (i)ROS1rearrangements are rare (1–2% of NS-NSCLC), (ii) the specificity of ROS1 IHC is not optimal, and (iii)ROS1FISH is not widely available, making this algorithm challenging to interpret time-consuming. We evaluated RNA NGS, which was used as reflex testing forROS1rearrangements in NS-NSCLC with the aim of replacing ROS1 IHC as a screening method. ROS1 IHC and RNA NGS were prospectively performed in 810 NS-NSCLC. Positive results were analyzed byROS1FISH. ROS1 IHC was positive in 36/810 (4.4%) cases that showed variable staining intensity while NGS detectedROS1rearrangements in 16/810 (1.9%) cases.ROS1FISH was positive in 15/810 (1.8%) of ROS1 IHC positive cases and in all positive ROS1 NGS cases. Obtaining both ROS1 IHC andROS1FISH reports took an average of 6 days, while obtaining ROS1 IHC and RNA NGS reports took an average of 3 days. These results showed that systematic screening for the ROS1 status using IHC must be replaced by NGS reflex testing.