We demonstrate an improved MALDI-TOF MS-based beta-lactamase assay platform for cefotaxime, a representative third-generation cephalosporins, resistant bacteria. Cefotaxime that was incubated with the lysates of E. coli exhibiting extended-spectrum beta-lactamase (ESBL) activity was modified by Girard’s reagent T to introduce permanent positive charge at the carboxyl functional groups of cefotaxime, resulting in dramatic advances on the ionization efficiency and quantitativeness during MS analysis. The method was validated with clinical isolates of a variety of ESBL-producing E. coli. In comparison with conventional UPLC system, it showed reliable data for distinguishing cefotaxime-resistant bacteria. This improved MS-based beta-lactamase activity assay platform would be utilized to more rapidly and accurately detect clinically relevant antibiotic resistant bacteria in clinical settings.