Plasmid-encoded beta-lactamases are a major reason for antibiotic resistance in Gram negative bacteria. These enzymes hydrolyze the beta-lactam ring structure of certain beta-lactam antibiotics, consequently leading to their inactivation. The clinical situation demands for specific first-line antibiotic therapy combined with a quick identification of bacterial strains and their antimicrobial susceptibility. Strategies for the identification of beta-lactamase activity are often cumbersome and usually lack sensitivity and specificity. The current work demonstrates that matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an ideal tool for these analytical investigations. Herein, we describe a fast and specific assay to determine beta-lactamase activity in bacterial lysates. The feasibility of the analytical read-out was demonstrated on a MALDI-triple quadrupole (QqQ) and a MALDI time-of-flight (TOF) instrument, and the results allow the comparison of both approaches. The assay specifically measures enzyme-mediated, time-dependent hydrolysis of the beta-lactam ring structure of penicillin G and ampicillin and inhibition of hydrolysis by davulanic acid for clavulanic acid susceptible beta-lactamases. The assay is reproducible and builds the basis for future in-depth investigations of beta-lactamase activity in various bacterial strains by mass spectrometry.