We present the first synthesis of β-lactam-derived haptens, leveraging the principles of diversity-oriented synthesis to discover compounds for drug allergy in vitro testing. We designed, synthesised, and performed in vitro immunological evaluation on 18 structurally diverse haptens derived from β-lactam antibiotics. The antigens obtained with the synthesised haptens allow for the detection of specific anti-β-lactam immunoglobulins G and E. Excellent diagnostic sensitivity (83%) and specificity (100%) were achieved when the panel of antigens was tested against a cohort of 31 human serum samples using a multiplexed compact disc-based in vitro testing tool. We posit that adopting this strategy could aid β-lactam delabeling initiatives.