Background: Pleural effusion is commonly observed in patients with advanced lung cancer and its molecular profiling has not been fully investigated. Cell-free (cf) DNA or RNA isolated from cancer patient’s plasma has allowed noninvasive tumor genome analysis, however, there are few study of detection and characterization of cfDNA or cfRNA in pleural effusions and its application in cancer patient treatment. Here we reported the utilization of cell free total nucleic acid (cfTNA) isolated from pleural fluid of late stage lung cancer patient with Next-generation sequencing (NGS) liquid biopsy assay-Oncomine™ Lung cfTNA Assay. The assay simultaneously query about 168 hotspots across ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, and TP53, CNV (MET), gene fusions (ALK, RET, ROS1) as well as MET exon 14 skipping which have been frequently identified in NSCLC. A comprehensive study of these mutations provides information for therapy decision, disease monitor as well as new drug development.