Reliable diagnosis of food allergy is dependent on the analytes used. Approaches based on well-defined individual molecules of either natural or recombinant origin are likely to replace those based on food extracts in the future. Therefore, a library comprising well-characterized authentic natural and recombinant allergens was formed within the EC funded IP EuroPrevall. The advantages and disadvantages of including either natural or recombinant proteins are summarized, together with inclusion criteria such as purity and the implications of both allergenic and nonallergenic impurities for the performance of diagnostic assays. In order to harmonize quality criteria of the individual food allergens included in the library, as suite of characteristics and associated methodologies used to define them, was agreed. The application of these methods and impact on the quality and performance of the final purified food allergens included in the EuroPrevall allergen library is discussed.