Almost 20 years after the publication of the first microbial sequences, today's world is growing in the direction of the post-genomic era with the understanding of transcriptomics and proteomics, which offer insight into cellular physiology. As the function of protein is associated with the phenotypic characters of an organism, the power to exceed the entire protein network of a microbial world has a huge impact on microbiology. Nowadays, mass spectroscopy (MS) has been extensively used in microbiology for the identification, characterization, and serotyping. In recent times, the large genome size, its complexity, and their study is a big task for the microbiologist. It is interesting to see how scientists examine microbial proteomics and analyze them. Proteomics has a key task to carry out in endeavors to develop far-reaching cell maps of biochemical procedures happening inside explicit microorganisms at given spatial and worldly focuses. Here we are going to discuss the methodologies for the identification of bacteria up to species level with great accuracy with the use of proteomes of bacterial pure culture. This chapter will also discuss the sample preparation and identification of a specific strain of bacteria for the liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the recent application of it.