A method of characterizing a population of at least one microorganism which comprises, before the caracterization, the preparation of a characterization zone of an analysis plate in order to carry out the at least one characterization, the said preparation comprising the following steps in succession: - a step consisting in providing an analysis plate for a characterization by means of the MALDI technique, the plate comprising at least one analysis zone carrying an antimicrobial agent; - a step of depositing the population of at least one microorganism onto said analysis zone in contact with the antimicrobial agent; - an incubation step, consisting in preserving the analysis plate under conditions and for a sufficient time to allow the antimicrobial agent and the microorganism that is present to interact; and - a step of depositing a matrix that is suitable for the MALDI technique onto the analysis zone, in which, the population of at least one microorganism deposited on the analysis zone, then, undergoes at least one ionization step by bombarding the analysis zone with a laser beam in accordance with the matrix-assisted laser desorption-ionization mass spectrometry technique known as MALDI, the said caracterizing comprising: • identifying the family, genus, or, as is preferable, the species of a population of a microorganism deposited on the characterization zone by carrying out a first analysis by MALDI type mass spectrometry corresponding to a first step of ionizing the characterization zone, and by using a first calibration for the analysis; and • determining the possible presence on the characterization zone of a population of a microorganism that is resistant to the antimicrobial agent by carrying out a second analysis by mass spectrometry using the MALDI-TOF technique corresponding to a second step of ionizing the same characterization zone, and by using a second calibration for the analysis. - a step consisting in providing an analysis plate for a characterization by means of the MALDI technique, the plate comprising at least one analysis zone carrying an antimicrobial agent; - a step of depositing the population of at least one microorganism onto said analysis zone in contact with the antimicrobial agent; - an incubation step, consisting in preserving the analysis plate under conditions and for a sufficient time to allow the antimicrobial agent and the microorganism that is present to interact; and - a step of depositing a matrix that is suitable for the MALDI technique onto the analysis zone, • identifying the family, genus, or, as is preferable, the species of a population of a microorganism deposited on the characterization zone by carrying out a first analysis by MALDI type mass spectrometry corresponding to a first step of ionizing the characterization zone, and by using a first calibration for the analysis; and • determining the possible presence on the characterization zone of a population of a microorganism that is resistant to the antimicrobial agent by carrying out a second analysis by mass spectrometry using the MALDI-TOF technique corresponding to a second step of ionizing the same characterization zone, and by using a second calibration for the analysis.