Tomeet clinical diagnostic needs and for general microbiologicalscreening, it is essential to be able to accurately and rapidly identifyany microorganisms from complex microbiota. To gain insight into theindividual components of microbiota, culturomics has been proposedas a means to systematically test hundreds of possible cultivationconditions and generate numerous microbial isolates with very distinctcharacteristics. High-throughput identification methods must now bedeveloped to quickly screen these isolates. Currently, most multiplexingmethods involve labeling, which comes at a cost. In this paper, wepresent an innovative label-free multiplexing method for the identificationof microorganisms using tandem mass spectrometry. The method is basedon offline reversed-phase fractionation of individual peptidomes.Multiplexing is achieved by mixing fractions of staged hydrophobicity;thus, each sample is mapped to specific elution times. In this proof-of-conceptstudy, multiplexed samples were analyzed by tandem mass spectrometryin a single run and microorganisms present in the mixture were resolvedby phylopeptidomics proteotyping. Using this methodology, up to 21microorganisms could be identified in a single 60 min run performedwith a Q-Exactive HF high-resolution mass spectrometer, resultingin a rate of one microorganism identified per 3 min of mass spectrometry,without any need for the use of labeling reagents. This approach opensnew perspectives for the application of high-throughput proteotypingof bacteria using tandem mass spectrometry in large culturomics projects.