Over the last two decades, numerous genomes of pathogenic bacteria have been fully sequenced and annotated, while others are continuously being sequenced. To date, the sequences of more than 8,500 whole bacterial genomes are publicly available for research purposes. These efforts in high-throughput sequencing simultaneously to progresses in methods allowing to study whole transcriptome and proteome of bacteria provide the basis of comprehensive understanding of metabolism, adaptability to environment, regulation, resistance pathways, or pathogenicity mechanisms of bacterial pathogens. Staphylococcus aureus is a Gram-positive human pathogen causing a wide variety of infections ranging from benign skin infection to life-threatening diseases. Furthermore, the spreading of multidrug-resistant isolates requiring the use of last barrier drugs has resulted in a particular attention of the medical and scientific community to this pathogen. We describe here proteomic methods to prepare, identify, and analyze protein fractions, which allow studying Staphylococcus aureus on the organism level. Besides evaluation of the whole bacterial transcriptome, this approach might contribute to the development of rapid diagnostic tests and to the identification of new drug targets to improve public health.