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Resistance against β-lactam antibiotics is a growing challenge for managing severe bacterial infections. The rapid and cost-efficient determination of β-lactam resistance is an important prerequisite for the choice of an adequate antibiotic therapy. β-Lactam resistance is based mainly on the expression/overexpression of β-lactamases, which destroy the central β-lactam ring of these drugs by hydrolysis. Hydrolysis corresponds to a mass shift of +18 Da, which can be easily detected by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Therefore, a MALDI-TOF MS-based assay was set up to investigate different enterobacteria for resistance against different β-lactam antibiotics: ampicillin, piperacillin, cefotaxime, ceftazidime, ertapenem, imipenem, and meropenem.