Rapid monitoring of real bacterial metabolic perturbations to antibiotics may be helpful to better understand the mechanisms of action and more targeted treatment. In this study, the real metabolic responses to antibiotic treatment in living bacteria were profiled rapidly by induced electrospray ionization mass spectrometry. Significant metabolic perturbations were profiled after antibiotic treatment compared with untreated bacteria. Similar and unique metabolic responses were observed with different antibiotic treatments. Further multivariable analysis was performed to determine significant metabolites as potential biomarkers. Moreover, different metabolic disturbances were detected for serial dilutions of antibiotic treatments. Overall, combined with induced electrospray ionization mass spectrometry, the rapid and real bacterial metabolic status caused by antibiotics was monitored, suggesting the potential application of our method in mechanism exploration and clinical diagnosis.