PURPOSE Patients with metastatic ROS1 fusion–positive non–small cell lung cancer (NSCLC) are effectively treated with entrectinib, a multikinase inhibitor. Whether serial targeted gene panel sequencing of cell-free DNA (cfDNA) can identify response and progression along with mechanisms of acquired resistance to entrectinib is underexplored. METHODS In patients with ROS1 fusion–positive NSCLC, coclinical trial plasma samples were collected before treatment, after two cycles, and after progression on entrectinib (global phase II clinical trial, ClinicalTrials.gov identifier: NCT02568267 ). Samples underwent cfDNA analysis using MSK-ACCESS. Variant allele frequencies of detectable alterations were correlated with objective response per RECIST v1.1 criteria. RESULTS Twelve patients were included, with best response as partial response (n = 9, 75%), stable disease (n = 2, 17%), and progressive disease (PD; n = 1, 8%). A ROS1 fusion was variably detected in cfDNA; however, patients without a ROS1 fusion in cfDNA had no other somatic alterations detected, indicative of possible low cfDNA shedding. Clearance of the enrolling ROS1 fusion or concurrent non- ROS1 alterations ( TP53, CDH1, NF1, or ARID1A mutations) was observed in response to entrectinib therapy. Radiologic PD was accompanied by redemonstration of a ROS1 fusion or non- ROS1 alterations. On-target resistance was rare; only one patient acquired ROS1 G2032R at the time of progression. Several patients acquired new off-target likely oncogenic alterations, including a truncating alteration in NF1. CONCLUSION Serial cfDNA monitoring may complement radiographic assessments as determinants of response and resistance to entrectinib in ROS1 fusion–positive lung cancers in addition to detecting putative resistance mechanisms on progression.