AbstractExhaled breath condensate (EBC) is a biological fluid that contains trace amounts of secreted pulmonary proteins, and is emerging as a potentially valuable and non‐invasively obtained source of disease biomarkers. Proteome analysis of these samples could lead to the identification of prognostic indicators of airway diseases. The objective of this study was to develop a protocol for proteome analysis of EBC samples. In this report, an improved procedure for EBC sample preparation and concentration is presented, together with a method for comparison of the protein profiles between two groups. The presented approach enabled to study the condensed exhaled breath proteome for biomarker analysis, and revealed proteins not previously identified in an EBC proteomics approach. In a comparative pilot study, EBC protein profiles obtained from smokers and non‐smokers showed distinct differences and are illustrative for its potential in clinical studies. EBC from smokers contained higher concentrations of the more abundant proteins, such as cytokeratins, compared to non‐smokers, and calgranulin B was identified uniquely in EBC samples from smokers.