The objective of this experimental study was to discover volatile metabolites present in exhaled breath that could be used as biomarkers of gastro-esophageal reflux disease, GERD, one of the most common causes of chronic cough. An in vitro model based on pork tissue samples exposed to a challenge by artificial gastric fluid was used to identify specific volatile compounds to be chosen for quantification in directly exhaled breath of GERD patients and controls using selected ion flow tube mass spectrometry, SIFT-MS. GC/MS analyses of the headspace of this in vitro model indicated that the only volatile compound significantly increased was acetic acid. End expiratory concentration of acetic acid measured by SIFT-MS in mouth exhaled breath of 22 GERD patients (median 85 ppbv) was found to be significantly higher than that in breath of a control group (median 48 ppbv). Breath acetic acid may be useful for non-invasive diagnostics of GERD and other conditions resulting in the lowering of pH of the lining of the airways.