Aims The identification and differentiation of antibiotic-resistant bacteria by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) profiling remains a challenge due to the difficulty in detecting unique protein biomarkers associated with this trait. To expand the detectable proteome in antibiotic-resistant bacteria, we describe a method implementing offline LC protein separation/fractionation prior to MALDI-ToF-MS and top-down MALDI-ToF/ToF-MS (tandem MS or MS/MS) for the analysis of several antibiotic-resistant Escherichia coli isolates. Methods and Results Coupling offline LC with MALDI-ToF-MS increased the number of detected protein signals in the typically analyzed mass regions (m/z 3000-20 000) by a factor of 13. Using the developed LC-MALDI-ToF-MS protocol in conjunction with supervised principal components analysis, we detected a protein biomarker at m/z 9355 which correlated to beta-lactam resistance among the E. coli bacteria tested. Implementing a top-down MALDI-ToF/ToF-MS approach, the prefractionated protein biomarker was inferred as a DNA-binding HU protein, likely translated from the bla(CMY-2) gene (encoding AmpC-type beta-lactamase) in the incompatibility plasmid complex A/C (IncA/C). Conclusions Our results demonstrate the utility of LC-MALDI-MS and MS/MS to extend the number of proteins detected and perform MALDI-accessible protein biomarker discovery in microorganisms. Significance and Impact of the Study This outcome is significant since it expands the detectable bacterial proteome via MALDI-ToF-MS.