In a time in which the spread of multidrug resistant microorganisms is ever increasing, there is a need for fast and unequivocal identification of suspect organisms to supplement existing techniques in the clinical laboratory, especially in single bacterial colonies. Mass-spectrometry coupled with efficient peptide separation techniques offer great potential for identification of resistant-related proteins in complex microbiological samples in an unbiased manner. Here, we developed a capillary electrophoresis-electrospray ionization-tandem mass spectrometry CE-ESI-MS/MS bottom-up proteomics workflow for sensitive and specific peptide analysis with the emphasis on the identification of β-lactamases (carbapenemases OXA-48 and KPC in particular) in bacterial species. For this purpose, tryptic peptides from whole cell lysates were analyzed by sheathless CE-ESI-MS/MS and proteins were identified after searching of the spectral data against bacterial protein databases. The CE-ESI-MS/MS workflow was first evaluated using a recombinant TEM-1 β-lactamase, resulting in 68% of the amino acid sequence being covered by 20 different unique peptides. Subsequently, a resistant and susceptible Escherichia coli lab strain were analyzed and based on the observed β-lactamase peptides, the two strains could easily be discriminated. Finally, the method was tested in an unbiased setup using a collection of in-house characterized OXA-48 (n = 17) and KPC (n = 10) clinical isolates. The developed CE-ESI-MS/MS method was able to identify the presence of OXA-48 and KPC in all of the carbapenemase positive samples, independent of species and degree of susceptibility. Four negative controls were tested and classified as negative by this method. Furthermore, a number of extended-spectrum beta-lactamases (ESBL) were identified in the same analyses, confirming the multiresistant character in 19 out of 27 clinical isolates. Importantly, the method performed equally well on protein lysates from single colonies. As such, it demonstrates CE-ESI-MS/MS as a potential next generation mass spectrometry platform within the clinical microbiology laboratory.