Proteomics-based identification strategy for gluten proteins in ethanolic extracts of wheat flour is described in this study. Protein extracts were separated by 1D gel electrophoresis and subjected to in-gel digestion with chymotrypsin which was found an appropriate proteolytic enzyme for the purpose. Based on collision-induced dissociation of selected chymotryptic peptides using MALDI QIT RTOF and MALDI TOF/TOF, the high-molecular-weight glutenin subunit DX5 and Α-gliadin were identified. This strategy allows unambiguous identification of proteins toxic for patients with coeliac diseases`.