Updated project metadata. Flucloxacillin is a β-lactam antibiotic associated with a high incidence of drug-induced liver injury. Although expression of HLA-B*57:01 increases susceptibility, little is known of the pathological mechanisms involved in the induction of the clinical phenotype. Irreversible protein modification is suspected to drive the reaction through the provision of flucloxacillin-modified peptides that are presented to T-cells by the protein encoded by the risk allele. In this study, the binding of flucloxacillin to human primary hepatocytes was characterized. Flucloxacillin was shown to bind to multiple intracellular proteins including major hepatocellular proteins (haemoglobin and albumin) and mitochondrial proteins (glutamate dehydrogenase and carbamoyl-phosphate synthase). Inhibition of membrane transporters multidrug resistance-associated protein 2 (MRP2) and P-glycoprotein (P-gp) appeared to reduce the levels of covalent binding. These flucloxacillin-modified intracellular proteins provide a potential source of neo-antigens for HLA-B*57:01 presentation by hepatocytes, that may drive CD8+ T-cell responses. More importantly, covalent binding to mitochondrial proteins, particularly those involved in the metabolism of both endogenous compounds and xenobiotics, could potentially affect their functional activity. This may increase cellular stress signalling and alter the regulatory threshold for activation of the adaptive immune system and thereby play an important role in determining the progression and severity of liver injury. Of particular interest, flucloxacillin binding to Lys50 on 14-3-3 proteins, a key residue within the 14-3-3 protein phospho-binding pocket, could potentially block its binding to signalling proteins as demonstrated by computational modelling, leading to regulating major cellular functions including cell proliferation, growth, apoptosis, autophagy, and cell motility.