The present invention relates to a method for characterizing the antibiotic resistance of a microorganism, the method comprising the steps of (a) providing a reference mass spectrum of an antimicrobial compound, its enzymatic modification product, its molecular target, or of a substrate compound of a its modifying enzyme; (b) exposing a microorganism, a cell lysate thereof, or a growth medium supernatant thereof, to the antimicrobial compound or the substrate compound in aqueous liquid to thereby provide an exposed sample; (c) acquiring a mass spectrum of the exposed sample; (d) comparing the mass spectrum acquired in step c) with the reference mass spectrum of step (a), and (e) determining from the comparison whether modification of the antimicrobial compound, its modification product or its molecular target or of the substrate has occurred following the exposure, and establishing that the microorganism is potentially resistant to the antimicrobial compound when the modification is observed.