〔Abstract〕 Fifty-four extended-spectrum β-lactamases(ESBLs) producing strains ofKlebsiella pneumoniaeand 24 cephalosporins-sensitive strains were randomly collected. The mass peaks of hydrolyzed antibiotics were detected by MALDI-TOF MS after co-incubation with a certain concentration of antibiotics. The hydrolysis assay of cefotaxime and ceftriaxone was able to identify ESBLs-producingKlebsiella pneumoniaeafter co-incubation for 1 h and 2 h respectively. Compared with genotype detection of ESBLs, the specificity was 100% and the sensitivity was 96.3%. MALDI-TOF MS can quickly and accurately identify pathogens in clinical practice, which has great potential in the detection of bacterial resistance. The antibiotic hydrolysis assay based on MALDI-TOF MS can detect ESBLs-producingKlebsiella pneumoniaewithin few hours.