Workflow for the preparation of tryptic digests of cell extracts from S. enterica subsp. arizonae, S. enterica subsp. enterica, and S. enterica subsp. houtenae and subsequent MALDI-TOF/TOF MS analysis. The cell extract used for classical biotyping is dried on a SpeedVac. After resolubilizing the pellet through the addition of RapiGest detergent under 5 min of high-intensity focused ultrasound (HIFU) treatment, trypsin is added and the proteins are digested for 15 min under the assistance of HIFU (in an ice water bath). The peptides are separated by nano-LC, mixed with HCCA matrix directly in the fraction collector, and spotted onto the MALDI target. MALDI-TOF/TOF analysis is performed, and the resulting data are screened for biomarker peptide candidates using an Excel macro developed in house.