The increase in multidrug-resistant microorganisms that produce extended-spectrum β-lactamases (ESBLs) and carbapenemases is a serious problem worldwide. Recently, matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) has been used for the rapid detection of antibiotic-resistant bacteria. The objective of this study was to establish a method to detect ESBL-producingEscherichia coliby monitoring the hydrolyzation of cefotaxime (CTX) using MALDI-TOF MS. According to the ratio of the peak intensity of CTX and hydrolyzed-CTX-related compounds, the ESBL-producing strains could be clearly distinguished after 15 min of incubation. Moreover, the minimum inhibitory concentration (MIC) values forE. coliwere 8 μg/mL and lower than 4 μg/mL, which could be distinguished after 30 min and 60 min of incubation, respectively. The enzymatic activity was determined using the difference in the signal intensity of the hydrolyzed CTX at 370 Da for the ESBL-producing strains incubated with or without clavulanate. The ESBL-producing strains with low enzymatic activity orblaCTX-Mgenes could be detected by monitoring the hydrolyzed CTX. These results show that this method can rapidly detect high-sensitivity ESBL-producingE. coli.