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Recently, it was speculated that human serum albumin (HSA) possesses β-lactamase activity and could contribute to nonmicrobial-based antibiotic resistance, owing to its ability to hydrolyse the β-lactam ring of nitrocefin. Moreover, the putative β-lactamase activity of HSA has been shown to increase significantly in the presence of environmental pollutants (1-naphthol and 2-naphthol). It was postulated that HSA could also cleave the β-lactam ring of clinically significant antibiotics. We studied the β-lactamase activity of HSA on clinically significant antibiotics of cephalosporin group in the presence of environmental pollutants by determining specific activity, enzyme kinetics and minimum inhibitory concentrations (MIC). The specific activity of HSA on various cephalosporins was found to be 1181–34 550 times lower than that observed for recombinant CTX-M-15 (used as positive control). The catalytic efficiency (kcat/Km) of HSA on nitrocefin hydrolysis was 126·7 times lower than that of recombinant CTX-M-15, and it has increased only 2- to 3-folds in the presence of environmental pollutants. Moreover, cephalosporins were not hydrolysed by HSA under experimental conditions. The MIC data also showed that HSA is incapable of hydrolysing cephalosporins. The study concludes that HSA is inefficient to cleave antibiotics of cephalosporin group and hence does not contribute to nonmicrobial-based antibiotic resistance.Significance and Impact of the Study: Earlier reports showed that human serum albumin (HSA) possesses β-lactamase activity, owing to its ability to cleave nitrocefin, and thus contributes to antibiotic resistance. Also, its β-lactamase activity is augmented when exposed to pollutants. As nitrocefin is not an antibiotic of clinical use, the conclusion drawn does not represent a true scenario and is misleading. Our results showed that HSA is inefficient in cleaving nitrocefin as compared to a true β-lactamase (CTX-M-15) and is practically inactive on cephalosporin antibiotics even in the presence pollutants. The findings showed that HSA-mediated hydrolysis of β-lactam antibiotics does not contribute to antibiotic resistance.