Background and aims: to assess the sensitivity and specificity for the basophil activation test (BAT) and the detection of drug-specific antibodies (IgE) in the retrospective diagnosis of β-lactam allergy by using assay- specific thresholds as resulted from the performance of receiver operating characteristics curve (ROC) analysis and to describe a sequential algorithm that might increase diagnostic sensitivity and reduce the number of patients that should undergo drug challenge tests by the joint use of in vivo and in vitro diagnostic tests. Methods: 37 patients with suspected β-lactam immediate-type hypersensitivity reactions were tested for the culprit drug. 31 healthy controls were similarly tested. BAT was performed with Flow2Cast technique (Bühlmann Laboratories, Switzerland). Drug-specific IgE antibodies were detected using “sandwich”-type radio-immunoassay (RIA) with sepharose as solid phase (Pathologie Université „H. Poincare”, France). Results: ROC curve analysis identifies an optimal threshold>1.9 for RIA positivity, with 70.3% sensitivity and 90.3% specificity. For BAT, an optimal threshold>1.97 yields 51.4% sensitivity and 90.3% specificity. 89.18% of the patients were diagnosed by the combined use of skin tests and in vitro tests results, while 10.82% of the patients had negative tests results. Conclusions: RIA seems to have higher sensitivity than BAT (70.3% versus 51.4%) for β-lactams. The joint use of allergy diagnostic tests has 89.18% sensitivity. A combination of allergologic skin tests, detection of antibiotic- specific antibodies, followed by BAT may be suitable for investigating β-lactam hypersensitivity reactions since no diagnostic test has absolute diagnostic accuracy.