We have previously demonstrated the analysis of intact proteins directly from bacterial colonies (including Gram-negative and Gram-positive clinical isolates) grown on agar media by liquid extraction surface analysis mass spectrometry (LESA MS). Several challenges were identified in that work, including (1) interference of background signal derived from the nutrient media ( Escherichia coli), (2) a high density of protein peaks leading to the isolation of multiple protein precursor ions in a single window and consequent acquisition of composite tandem mass spectra ( Pseudomonas aeruginosa), and (3) the overabundance of secreted peptides suppressing peaks corresponding to proteins ( Staphylococcus aureus). Here, we present the coupling of high-field asymmetric waveform ion mobility spectrometry (FAIMS) separation into the LESA MS protocol, with the aim of resolving the aforementioned challenges and thus improving the capabilities of LESA MS for bacterial characterization. The results show that inclusion of FAIMS expands the range of detected proteins through separation of background peaks from protein signal, as well as through resolution of overlapping protein peaks which could not previously be isolated by LESA MS alone.