Sample preparation is the most crucial step in the proteome research of microbes. 2D gel electrophoresis (2-DGE) is a high throughput approach by which all proteins imprint on the gel in the form of spots or dots. Various protocols for the extraction of proteins exist in the literatures that are compatible with 2-DGE based proteome analysis from different microbes. Analysis of low abundance proteins, the solubility of proteins, and their resolution on the gel are some major issues observed with sample preparation and 2-DGE. To combat these issues, researchers have developed improved versions of the existing protocols using detergent/chaotropes for the enrichment of proteins during extraction along with the compatible chemical precipitation for better resolution of 2D gel pattern. In this chapter, we will discuss the overview, challenges encountered during the protein sample preparation, and their possible solutions in order to get a better 2-D gel of microbial proteins.