We report a case of anaphylaxis caused by cloxacillin in a 13-year-old patient. The basophil activation test, performed 25 days after the anaphylactic reaction, was positive to cloxacillin, amoxicillin, and penicillin G and negative to ibuprofen, tolerated by the patient. The analysis was performed 17 days after the reaction was not conclusive because 74% of the basophil population was activated in basal conditions. The abnormally high activation was similar to that found in an analysis before the reaction, exactly 4 days after finishing a well-tolerated treatment with amoxicillin. This first analysis was available because a patient's sample was taken from the emergency laboratory as a blind control for a study to assess the basophil activation test reliability in diagnosis of hypersensitivity to NSAIDs. The high number of activated basophils in basal conditions after treatment with amoxicillin and before the anaphylactic reaction to cloxacillin probably reflects the beginning of the sensitization. Until now, no cases of hypersensitivity to cloxacillin have been diagnosed by means of the basophil activation test.