Food allergy is a widespread phenomenon: An estimated 1–2% of the population suffers from some type of food allergy. Fruits and vegetables are considered among the most important elicitors of food allergy. Berry fruits rich in phenolic compounds including flavonols, flavones and anthocyanins have recently gained increasing interest due to their possible beneficial effects on human health. However, they may harbour a series of allergenic proteins that cause discomfort or even represent serious threats to certain individuals. The identification and characterization of allergens in fruits like berries from distant taxa, requires novel approaches involving genomic and proteomic tools. The allergen content of blueberries (Vaccinium myrtillus), strawberries (Fragaria ananassa), raspberries (Rubus idaeus), and blackberries (Rubus fruticosus) was determined. Different extraction procedures are required to improve protein detection with polyclonal and monoclonal antisera raised against different allergens from Malus domestica in Western blotting. Due to the cross-reactivity they are able to recognize similar epitopes in highly conserved protein families of other plant species. Fruit extracts were analysed by two dimensional electrophoresis and mass spectrometry. Using clinical screening tools from a high number of patients and patient sera delivered conclusively results on allergenic proteins present in the small fruits.