Wheat belongs to six major food allergens inducing IgE-mediated hypersensitivity reaction manifesting as cutaneous, gastrointestinal, and respiratory symptoms. Although cereals are a staple food item in most diets, only a few wheat proteins causing hypersensitivity have been identified. To characterize wheat allergens, salt-soluble wheat extracts were separated by 1-DE and 2-DE and IgE-binding proteins were detected by immunoblotting using sera of patients with allergy to ingested wheat. Proteins, frequently recognized by IgE on 2-DE were analyzed by MALDI-TOF and QTOF and their spectrum was completed by 1-DE and LCQDECA nLC-MS/MS IT technique. Using all three techniques we identified 19 potential wheat allergens such as α-amylase inhibitors, β-amylase, profilin, serpin, β-D-glucan exohydrolase, and 27K protein. Employing newly developed ELISA, levels of IgE Abs against Sulamit wheat extract and α-amylase inhibitors type 1 and 3 were quantified and shown to be significantly elevated in sera of allergic patients compared to those of healthy controls. The level of IgE Abs against α-amylase inhibitor type 3 was lower, slightly above the cut-off value in the majority of patients' sera. Our findings contribute to the identification of wheat allergens aimed to increase the specificity of serum IgE and cell activation diagnostic assays.