In the field of allergy diagnosis, most in vitro functional tests are focused on basophils. Nevertheless, the very small number of circulating basophils limits these experiments and their clinical benefit remains controversial. As flow cytometry is a valuable tool for identifying cell populations, even at low concentrations, we developed a tricolour flow cytometric method for the study of allergen-induced basophil activation. Identification of cells was based both on CD45 expression and on the presence of IgE on the cell surface, since basophils express high-affinity receptors for IgE (FcεRI). Cell activation upon allergen challenge was assessed by the expression of CD63 antigen on the plasma membrane. Basophil isolation and activation (with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine) were validated in 32 non-allergic patients. In 12 allergic patients, basophil stimulation by a relevant allergen was in most cases positive (10/12). Furthermore a concentration-dependent hook effect was observed. Of the allergic and non-allergic patients, none showed non-specific activation with an irrelevant allergen (specificity 100%). Overall, our preliminary results, even in a small population, suggest that this is a reliable and valuable method for the diagnosis of allergies complementing specific allergen IgE and skin test results. Obviously, additional clinical studies are needed to validate these first results.