The membranes of eukaryotic cells contain microdomains that are different in lipid composition to the surrounding membrane and aggregate a number of proteins related to signal transduction and protein trafficking. These are referred to as lipid rafts or membrane rafts and are specialized in the regulation of cellular processes related to signal transduction, protein sorting, and membrane trafficking. The integrity of lipid rafts is important for the correct functionality of these raft-harbored cellular processes, and their alteration is related to the occurrence of severe diseases. We recently discovered that the membranes of bacterial cells also organize their signal transduction pathways in functional membrane microdomains that are structurally and functionally similar to the lipid rafts of eukaryotic cells. The existence of lipid rafts in the membrane of bacteria suggests that bacteria are more complex organisms than previously appreciated, and thus, their cellular complexity should be explored in more detail. In this protocol, we provide a detailed description of the materials and techniques that are necessary to purify the lipid rafts from bacterial membranes, which is a necessary step to explore the number of proteins and lipid species that constitute these membrane platforms. This is an essential protocol for any laboratory interested in exploring any aspect related to organization of lipid rafts in bacterial membranes.