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Background: Diagnostic tests for allergy rely on detecting allergen‐specific IgE. Component‐resolved diagnostics incorporate multiple defined allergen components to improve the quality of diagnosis and patient care. Objective: To develop a new approach for determining sensitization to specific allergen components that utilizes fluorescent protein tetramers for direct staining of IgE on blood basophils by flow cytometry. Methods: Recombinant forms of Lol p 1 and Lol p 5 proteins from ryegrass pollen (RGP) and Api m 1 from honeybee venom (BV) were produced, biotinylated, and tetramerized with streptavidin‐fluorochrome conjugates. Blood samples from 50 RGP‐allergic, 41 BV‐allergic, and 26 controls were incubated with fluorescent protein tetramers for flow cytometric evaluation of basophil allergen binding and activation. Results: Allergen tetramers bound to and activated basophils from relevant allergic patients but not controls. Direct fluorescence staining of Api m 1 and Lol p 1 tetramers had greater positive predictive values than basophil activation for BV and RGP allergy, respectively, as defined with receiver operator characteristics (ROC) curves. Staining intensities of allergen tetramers correlated with allergen‐specific IgE levels in serum. Inclusion of multiple allergens coupled with distinct fluorochromes in a single‐tube assay enabled rapid detection of sensitization to both Lol p 1 and Lol p 5 in RGP‐allergic patients and discriminated between controls, BV‐allergic, and RGP‐allergic patients. Conclusion: Our novel flow cytometric assay, termed CytoBas, enables rapid and reliable detection of clinically relevant allergic sensitization. The intensity of fluorescent allergen tetramer staining of basophils has a high positive predictive value for disease, and the assay can be multiplexed for a component‐resolved and differential diagnostic test for allergy. [ABSTRACT FROM AUTHOR]