In this study, the protein and peptide fractions of two commercial Italian barley malt beers, made with different processes by the same producer, have been analysed with a combined immunochemical and mass spectrometry approach. The “gluten” content of beer samples, measured with the R5 monoclonal antibody, was below the caution limit proposed by the Codex Alimentarius for gluten-free foods. The proteomic approach allowed to identify a 17 kDa avenin-like protein partially homologous to hordeins, that was particularly abundant in foam, in addition to the already reported barley albumins (Z 4-barley and ns-LTPs) and to minor amounts of yeast glycolytic enzymes. No intact hordeins were detected, although fragments derived from γ 3- and B-hordein were present. In consideration of the many implications of the protein/peptide pattern, these data provide useful information to improve quality and safety of beer.