Background: Leptomeningeal disease (LMD) is associated with significant morbidity and mortality for metastatic non-small cell lung cancer (mNSCLC). The standard of care for diagnosing LMD remains magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) cytology, but these have limited sensitivity. CSF-based liquid biopsy is an emerging strategy for dynamic assessments of CSF-derived tumor cells to monitor intracranial responses and CNS clonal evolution during treatment and at progression. However, data for the feasibility and utility of this approach is limited. We describe our clinical experience to evaluate the use of molecular analysis on CSF-derived circulating tumor cells (CTCs) for mNSCLC. Methods: Patients with mNSCLC who had a lumbar puncture for CSF collection as part of routine clinical care to rule out LMD were included in the study. CSF was evaluated for CTCs and cell-free DNA (cfDNA) with a commercially available assay (CNSide) and subsequent molecular profiling was performed to detect actionable mutations. Patients were analyzed for LMD with CSF cytology and MRI imaging. Molecular testing results from sequencing of tumor tissue and plasma cell tumor DNA were available for all patients. Other than copy number amplification detection for MET, the CSF assay allowed for analysis of MET and HER2 expression via fluorescent in situ hybridization (FISH). Results: The study included 22 patients with mNSCLC (77% female; median age 60 years; 95% adenocarcinoma). Sensitizing EGFR mutations were found in 78% of patients and 18% had an atypical EGFR mutation at initial diagnosis. LMD was diagnosed using the CSF CTC assay in thirteen of the 22 patients (59%) patients. CSF cytology was negative for LMD in five of 13 cases (38%) and furthermore, 2 of these cases (15%) had normal MRI brain imaging. There were sufficient CTCs to perform molecular profiling on seven of the 13 patients (54%). The concordance with tissue NGS was 100% and the driver mutation was identified in all 7 patients with the CSF CTC assay. MET expression and HER2 expression via FISH were noted in 11 patients (50%) and 4 patients (18%) respectively. Conclusions: In this single institution study, we detected a higher sensitivity to diagnose LMD with CNSide. This CSF assay identified 38% of LMD cases which were missed by standard CSF cytology and of note, 15% of the cases found with LMD were missed by both CSF cytology and MRI. In addition, CSF molecular testing using this assay demonstrated high concordance with tissue-based molecular testing. The study illustrates that multigene molecular analysis of CSF CTCs can be a useful tool for diagnosis, monitoring of response to therapy, as well as for the identification of therapeutic targets specific to the CSF especially for patients with oncogene-driven mNSCLC.