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Proteomics approaches combine high resolution two-dimensional separation techniques such as isoelectric focusing and gel electrophoresis or chromatography with mass spectrometry (MS) followed by database searching. Powerful bioinformatics tools are required to extract the relevant data from the complex mass spectra, predict protein/peptide masses, isotopic patterns, enzymatic cleavage sites and possible modifications like ammonia loss, water loss or oxidation. Trace amounts of gluten and food allergens are most commonly analyzed using enzyme-linked immunosorbent assays (ELISAs) that are specific, sensitive, comparatively cheap, easy to handle and suitable for routine analyses. However, ELISAs may face difficulties in detecting the target antigen in foods that have been extensively processed, hydrolyzed or fermented or that contain a variety of possibly interfering substances, such as polyphenols. Due to its versatility, sensitivity, selectivity and wide applicability, MS may serve as an independent reference method, especially for foods where ELISA results typically have low recoveries. After appropriate extraction of gluten and allergens from the food matrix and enzymatic digestion, the resulting peptide mixture is purified and separated by liquid chromatography prior to the detection and quantitation of characteristic marker peptides. In case of gluten, this workflow has been applied to the detection of wheat flour in oat flour, the relative quantitation of gluten in a variety of beers and to identify wheat varieties with low immunogenic potential. One major difficulty is to relate the amount of peptides back to the original protein content to comply with legislation. Due to the expensive equipment and expertise required, proteomics approaches are limited to specialized laboratories, but allow the simultaneous detection of gluten and multiple allergens in one run.