Tandem mass spectrometry-based proteotyping of microorganisms presents several advantages over whole-cell MALDI-TOF mass spectrometry: because a larger number of signals are recorded with better accuracy and precision, the approach allows for the identification of microorganisms at more resolved taxonomic levels, and can easily manage complex samples. Additionally, the use of SP3 paramagnetic beads for cell lysis and protein cleanup simplifies sample preparation for proteotyping. Based on these features, we have developed and tested a 96-well plate platform for high-throughput proteotyping of a large variety of bacteria. We evaluated the performance of the platform in terms of bacterial load and found no cross-contamination between wells. Likewise, phylopeptidomics analysis revealed no alteration in the relative quantifications of microorganisms. Finally, we applied this new format for rapid proteotyping of a large set of dental isolates using double-barrel chromatography coupled to tandem mass spectrometry, which maximizes the number of spectra per unit of time. The procedure allowed us to establish whether these isolates were pure strains or mixtures of strains and to identify the microorganisms at the most resolved taxonomic level.Significance: The rapid and accurate identification of microorganisms is a clinical priority in medical diagnostics; however, specimens containing mixtures of microorganisms are difficult to analyze and the procedure is time-consuming. Tandem mass spectrometry proteotyping allows the fast identification of complex mixtures of microorganisms, known or unknown, and can also establish the biomass ratio of each component. We describe here a new workflow for preparing microbial samples in a 96-well-plate format for tandem mass spectrometry proteotyping and document its advantages and limitations. We demonstrate that this new format coupled to a highly efficient double-barrel LC-MS/MS system allows proteotyping of 96 isolates in 55 h.