Carbapenems are broad spectrum antibiotics considered as a "last resort" medicine to treat bacterial infections. Carbapenem-hydrolyzing β-lactamases (also called carbapenemases), however, can confer bacterial resistance and represent a serious health threat. Here, we report a novel approach using (18)O labeling and selected reaction monitoring to detect carbapenemase activity from pathogenic microorganisms in a rapid and quantitative manner. Four model bacterial strains bearing various classes of β-lactamases were tested for their capability to hydrolyze Meropenem, an FDA-approved carbapenem drug. We were able to predict the Meropenem resistance of these bacteria on the basis of their carbapenemase activity, suggesting the great potential of our method in clinical diagnostics.