This chapter demonstrates a step-by-step and detailed protocol for isolating and culturing methanogenic endosymbionts which are crucial organisms in aid of digestion and methane synthesis in different niches. The first step of the approach is sample collection from anaerobic habitats through the enumeration technique and then the selection of specialized culture media that favors the methanogenic genera while adversely affecting other microbes. In this book chapter, we have described general methods of maintaining an anaerobic environment for the methanogens such as the use of gas-tight containers and a specific gaseous environment to enhance their growth. Key characteristics of these organisms’ isolation and long-term cultivation include the temperature, pH, nutrient content, and the need for some trace elements. Thus, this chapter intends to provide a reference to the investigators concerned with the ecological and biotechnological aspects of methanogenic endosymbionts. The isolation and maintenance of these microorganisms could be utilized effectively in the sustainable disposal of wastes, generation of renewable energies, and replenishment and conservation of the environment.