Next-generation shotgun proteomics is one of the most valuable tools for gaining insight into the function of organisms. By providing a list of peptides and abundance information, proteomics enables the identification of proteins, their quantities, posttranslational modifications, and localization. The most refined shotgun proteomics workflow involves protein extraction, trypsin digestion, ultrahigh-performance liquid chromatography coupled to high-resolution tandem mass spectrometry, and confident assignment of resulting spectra to peptide sequences. In this study, we present a versatile, time- and cost-efficient experimental workflow for protein extraction, digestion, and analysis that can be applied to any type of microorganism. Our experimental procedure exhibits superior sensitivity compared to gel-based protocols and can be used for comparative microbial proteomics to highlight key players that explain phenotypic differences between conditions or for proteotyping new microbial isolates for taxonomic purposes.