Background: Basophil activation tests (BATs) have promise for research and for clinical monitoring of patients with allergies. However, BAT protocols vary in blood anticoagulant used and temperature and time of storage before testing, complicating comparisons of results from various studies. Objective: We attempted to establish a BAT protocol that would permit analysis of blood within 24 hours of obtaining the sample. Methods: Blood from 46 healthy donors and 120 patients with peanut allergy was collected into EDTA or heparin tubes, and samples were stored at 4 degrees C or room temperature for 4 or 24 hours before performing BATs. Results: Stimulation with anti-IgE or IL-3 resulted in strong upregulation of basophil CD203c in samples collected in EDTA or heparin, stored at 4 degrees C, and analyzed 24 hours after sample collection. However, a CD63(hi) population of basophils was not observed in any conditions in EDTA-treated samples unless exogenous calcium/magnesium was added at the time of antiIgE stimulation. By contrast, blood samples collected in heparin tubes were adequate for quantification of upregulation of basophil CD203c and identification of a population of CD63 hi basophils, irrespective of whether the specimens were analyzed by means of conventional flow cytometry or cytometry by time-of- flight mass spectrometry, and such tests could be performed after blood was stored for 24 hours at 4 degrees C. Conclusion: BATs to measure upregulation of basophil CD203c and induction of a CD63 hi basophil population can be conducted with blood obtained in heparin tubes and stored at 4 degrees C for 24 hours.