Wheat gluten is a widely used ingredient in the food industry due to its unique properties and relatively low price. Modification of wheat gluten makes it a versatile ingredient and, thus, increases its applicability in foods. Therefore, gluten proteins can be found in unexpected sources, and this makes the gluten-free diet challenging to follow. Deamidation is one way to modify protein structure. It increases solubility and surface activity of gluten improving its functionality, but consequently, also influencing the accuracy of quantification by immunoassays. In this study, the effect of deamidation on the antibody recognition with gluten analysis methods based on monoclonal R5, omega-gliadin or G12 antibodies was studied. Random deamidation decreased the intensities to 13–54% of the intensity obtained for the intact peptides. Deamidation representing the transglutaminase deamidation decreased the intensities to 4–8%. Deamidation of gluten proteins abolished the recognition by omega-gliadin and G12 antibodies and decreased the recognition of R5 by 600 times when analyzed by the sandwich method and 125 times by the competitive method. In conclusion, with all of the investigated gluten-specific antibodies, deamidation decreased the affinity of antibodies to gluten peptides and proteins, which needs to be considered when assays and regulations are developed for gluten-free products.