|The growing number of antibiotic resistant bacteria is an increasing health care problem. The rapid detection and determination of resistant microorganisms are important for the application of the correct therapy. Resistance against ß-lactam antibiotics is characterized by the hydrolysis of the ß-lactam ring resulting in a mass shift of additional 18 Dalton. Since mass spectrometry is predestined to determine such mass differences an MALDI-TOF MS based assay was set up to analyze the hydrolysis of different ß-lactam antibiotics by the bacteria to be tested. Starting from single colonies, this novel approach allows the detection of resistance within 4 hours.