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The aim of this thesis was to determine activity of carbapenemases in Pseudomonas spp. isolates using mass spectrometry. There was created a new MALDI-TOF software based on Bruker‘s recommendations, which was able to detect activity of carbapenemases. Mass spectra were analyzed according to Bruker‘s procedure: there was been searching for the specific mass peaks which corresponded to the molecular weight of different carbapenem molecular forms. According to these values bacteria were assessed as sensitive (non-producing carbapenemases) and resistant (producing carbapenemases). During study period in 2015–2017 there was collected 165 carbapenem resistant P. aeruginosa isolates and 34 of them (20,61%) were producing hydrolyzing enzymes. Most of microorganisms were isolated from urine and wound exudate, less from stool and bronchial aspirate. The results also showed that majority of patients with carbapenem resistant P. aeruginosa strains were treated in units of intensive care (52,94%) and surgery (32,35%). More than a half of patients (70,59%) were men. Furthermore, the highest risk to be infected with P. aeruginosa had patients, who were over 60 years old. It was found that results of this study depended on lots of factors: concentration of antibiotic and its dilution time; chemical properties of carbapenems; composition of matrix, incubation and calibration solutions. It is important to note that MALDI-TOF mass spectrometer could not detect all the mechanisms of resistance in P. aeruginosa, so they could interfere detection of carbapenemases. Despite all the advantages of MALDI-TOF mass spectrometry in detection of carbapenemases (it lasts about 2 hours and it is easy to analyse mass spectra), this method has not being used in diagnostics yet.