Gluten is the collective name for a class of proteins found in wheat, rye, barley and oats. Coeliac disease (CD) is an immune-mediated inflammatory disease of the small intestine in a subset of genetically-susceptible individuals that is triggered by the ingestion of gluten, resulting in intestinal inflammation and damage. The only current treatment for CD- and gluten-intolerants (~70 million people globally) is lifelong avoidance of dietary gluten. Gluten-free (GF) foods are now commonplace, however, it is difficult to accurately determine the gluten content of GF-products using current methodologies as the antibodies are non-specific and show cross-reactivity. In processed products measurement is further confounded by protein modifications and/or hydrolysis. Global proteomic analysis of 14 economically important barley varieties utilising discovery LC-MS/MS was used to characterise the “gluteome” and select peptide markers specific to barley gluten (hordeins) and/or as general markers of barley for targeted quantitative MS assays. Barley-specific peptide markers were detected across 14 barley varieties with nine potential markers showing detection in all cultivars tested. The panel of barley peptide markers were then assessed against 16 commercial grains including those grown in crop rotation or typically processed in the same facility. Of these, seven of the peptide markers were unique to barley and can be used to develop sensitive assays for the presence of barley as an unlabelled food ingredient and show promise for the quantification of gluten to protect consumers suffering from CD.