Background: Approximately 10% of the global population identify themselves as penicillin allergic, yet 90% are not truly allergic and could safely tolerate penicillin. There is no simple way to identify these people. Current in vitro diagnostics include specific immunoglobulin E, sIgE (with a sensitivity of 19% and specificity of 97%) and basophil activation testing (BAT) with undefined sensitivity and specificity. Objective: To define the sensitivity and specificity of BAT in the diagnosis of penicillin allergyMethods: PubMed and EMBASE searched from inception to 04/02/2023 for original studies evaluating the performance characteristics of basophil activation test for penicillin allergy in adults. Study selection, data extraction, risk of bias, assessment with QUADAS-2 tool, certainty assessment with GRADE methodology were performed independently, in duplicate. Meta-analysis was performed using Reitsma methodology.Results: Twenty-two studies fulfilled the inclusion criteria. Twelve used the same positive threshold giving a summary point sensitivity 51% (95% CI, 46%-56%) and specificity 89% (95% CI, 85%-93%). Significant risk of bias was identified due to patient selection. GRADE certainty of evidence rated sensitivity “very low” due to imprecision and specificity as “low”. There was great heterogeneity in methods used. Use of 1000 basophils per test did not improve performance above 500 basophils. Conclusion: BAT sensitivity is highly variable across studies and remains too low to be considered as a routine element of clinical practice. BAT specificity is not as good as sIgE in penicillin allergy diagnosis. Significant further work is required in this field before clinical application of BAT in routine practice.