Amoxicillin-clavulanate (AC) is one of the most common causes of drug induced liver injury (DILI). The association between AC-DILI and HLA alleles and the detection of drug-specific T cells in patients with AC-DILI indicate that the adaptive immune system is involved in the disease pathogenesis. In this study, mass spectrometric methods were employed to characterize the antigen formed by AC in exposed patients and the antigenic determinants that stimulate T cells. Amoxicillin formed penicilloyl adducts with lysine residues on human serum albumin (HSA) in vitro, with K190 and K199 being the most reactive sites. Amoxicillin-modified K190 and K199 have also been detected in all patients, and more extensive modification was observed in patients exposed to higher doses of amoxicillin. In contrast, the binding of clavulanic acid to HSA was more complicated. Multiple adducts were identified at high concentrations in vitro, including those formed by direct binding of clavulanic acid to lysine residues, novel pyrazine adducts derived from binding to the degradation products of clavulanic acid, and a cross-linking adduct. Stable adducts derived from formylacetic acid were detected in all patients exposed to the drug. Importantly, analysis of hapten-protein adducts formed in the cell culture medium revealed that the highly drug-specific T-cell responses were likely driven by the markedly different haptenic structures formed by these two drugs. In this study, the unique haptenic structures on albumin in patients formed by amoxicillin and clavulanic acid have been characterized and shown to function as chemically distinct antigens which can stimulate separate, specific T-cell clones.