There is a need for non-invasive airway-based biomarkers in lung carcinogenesis for both risk assessment, and earlier diagnosis. Exhaled breath condensate (EBC) contains airway lining fluid molecules, including small molecules of polar and non-polar lipid origin, presumably in part from epithelial cellular origins. Here we pilot a GC-MS strategy for measurement of small, polar molecules in exhaled breath condensate in EBC from lung cancer patients and controls. Exhaled breath condensate (EBC) was collected non-invasively, using a handheld device (RTube) in ambulatory subjects engaged/recruited/consented through our pulmonary and thoracic surgery practices, under IRB protocol. A volume of 50 ul of EBC samples were combined with 200uL of methanol, containing 2 internal standards, 1nmol U13_succinate, 5nmol U13_citrate. Then, the samples were vortexed and 240 μl of supernatant was transferred to a sampling vial. The samples were dried under gentle nitrogen flow and derivatized with a two-step derivatization procedure, including a methoxyamine step for 90 minutes, and a silylation step for 60 minutes. QC sample was run multiple times during the analysis. The samples were analyzed by gas chromatography time-of-flight mass spectrometry (GC-TOFMS premier, Waters, USA). A number of 282 variables were detected after alignment and excluding any known artificial peaks, 49 of them were annotated. The data was normalized to the intensity of the sum of all the metabolites. The data set was then imported into SIMCA-p software (Umeå, Sweden) for multivariate analysis. A multivariate case-control ROC discriminant analysis compared the clinical model (AUC 0.72) to clinical plus exhaled small polar discriminant metabolites combined (AUC 0.92), showed incremental discrimination attributable to these metabolites (p=2.22e-62). ROC comparing clinical model (AUC 0.72) to clinical model plus metabolites (AUC 0.92); the difference attributable to metabolites was significant (p=2.22e-62). This exhaled biomarker platform can yield case-control discriminant small polar molecule sets related to known metabolic pathways, some of which are known to be deranged in cancers. Once further distilled and validated, our goal is to apply this non-invasive biomarker approach to prospective cohorts for non-invasive lung cancer risk assessment of the at-risk epithelium, in order to better select higher risk individuals to undergo effective CT screening. Supported by NIH-R21 CA192168-01; DoD-CDMRP- LC150738, NIH-NCI P60DK020541.