Background: Penicillin allergy delabeling initiatives are now part of antibiotic stewardship programs and include the use of invasive and risky in vivo tests. Instead, the quantiﬁcation of speciﬁc IgE is highly useful to conﬁrm immediate allergy to penicillins. However, discrepant results associated to the low sensitivity of the in vitro tests have limited their routine diagnostic use for delabeling purposes. We aimed to tackle a novel diagnostic strategy for specific IgE testing based on a homologous interpolation scheme, using recombinantly produced standards. Methods: Serum samples from a cohort of allergic patients and controls were analysed by a chemiluminescence-based immunoassay, using a bispecific binanobody as standard. The novel standard targets the major antigenic determinant of penicillin G and the paratope of Omalizumab, acting as human-like specific IgE. Results: Testing a cohort of 65 human serum samples, the method achieved a good agreement and strong positive relationship, reaching a limit of detection below 0.1 IU/mL. The sensitivity of the in vitro test significantly increased (66 %), doubling that of the ImmunoCAP reference in vitro assay with an overall specificity of 100 %. Conclusions: The new diagnostic strategy compares favourably with the results obtained on the ImmunoCAP system, paving the way towards the standardization of penicillin allergy testing. The recombinant standards are potent calibrators, highly stable, easy and inexpensive to produce, and overcome the limitation associated with preparations derived from pooled human serum, expediting the production of next generation standards with different specificities to successfully tackle β-lactam allergy delabeling by in vitro tests.