Protein extraction is a critical step in attaining optimal results in proteomic studies. Total protein extracts from grains such as wheat, barley, rye and oats can contain non-protein compounds that may interfere with the analysis of the proteome. To temper the effects of interfering compounds, strategies such as defatting and/or protein precipitation are often employed. In this study we have used LC-MS/MS experiments comparing six total protein extraction protocols that employed different buffer compositions, with and without defatting and protein precipitation steps, in order to profile two cultivars of each of the gluten-containing grains. Using Tris-HCl and urea-based buffers 1433 and 1769 proteins were extracted from the barley cultivar Sloop respectively. Inclusion of a hexane-based defatting step prior to protein extraction was of no benefit, whilst protein precipitation employed post-extraction negatively impacted protein recovery, a likely result of problems associated with protein re-solubilisation. The use of an alcohol-based extraction protocol commonly used for enriching for gluten proteins also yielded a lower number (645) of proteins but was noted to co-extract the alpha-amylase/trypsin inhibitors (ATIs) that have been implicated as elicitors of the poorly characterised condition non-Coeliac gluten sensitivity. Using the discovery data collected, we developed multiple reaction monitoring (MRM) MS assays for the ATIs derived from wheat, barley, rye and oats. These methods were applied to the quantitative determination of the optimal extraction protocol for ATI enrichment. A modified two-step extraction method, i.e. alcohol-based extraction followed by urea buffer extraction was shown to yield the maximum level of ATIs. The level of ATIs was noted to vary between cultivars, with LC-MRM-MS analysis providing an opportunity to select grain varieties with differing levels of ATIs.