The aim of the work was to test a relatively simple proteomics approach based on phenol extraction and two-dimensional gel electrophoresis (2-DE) with 7 cm immobilized pH gradient strips for the determination of clinically relevant proteins in wheat grain. Using this approach, 157 2-DE spots were quantified in biological triplicate, out of which 55 were identified by matrix-assisted laser desorption/ionization – time of flight tandem mass spectrometry. Clinically relevant proteins associated with celiac disease, wheat dependent exercise induced anaphylaxis, baker’s asthma, and food allergy, were detected in 24 2-DE spots. However, alcohol-soluble gliadins were not detected with this approach. The comparison with a recent quantitative study suggested that gel-based and gel-free proteomics approaches are complementary for the detection and quantification of clinically relevant proteins in wheat grain.