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Consumers, especially those with allergies and/or intolerances, should have confidence in two critical areas of food safety: foods should be correctly labelled and free from contamination. Coeliac disease is a condition that affects about one percent of the population, while non coeliac gluten sensitivity may affect up to a further 10% of the population. There is no current treatment for either of these conditions other than strict adherence to a life-long gluten-free diet. To ensure these diets are gluten-free it is critical to understand protein composition of gluten-containing grains, wheat, barley and rye, as well as oats for some sufferers of coeliac disease. This knowledge can then be used to develop quantitative markers for detection of the presence/absence of these cereals and for the specific proteins that comprise gluten from each species. These markers must be robust enough to not only work using the native cereals, but also when the samples are processed foods. Here we present the comprehensive characterisation, using mass spectrometry, of wheat and barley focussing on the “gluteome”. In addition we present a comparison of MRM-MS, the gold standard for peptide quantification to the relatively new data-independent acquisition strategy known as SWATH (sequential window acquisition of all theoretical spectra) MS.