This chapter elucidates the methodologies for the screening and characterization of biosurfactants produced by endosymbiotic microorganisms. Endosymbionts are defined as microorganisms residing within the tissues or cells of host organisms and have emerged as potential reservoirs for novel biosurfactants. We delineate cultivation techniques, screening assays, and analytical methods for the identification and characterization of biosurfactants synthesized by these endosymbionts. The methodology encompasses the isolation of endosymbionts from host tissues, preparation of appropriate growth media, and implementation of various screening techniques, including oil displacement assays, emulsification index determination, and surface tension measurements. Furthermore, we discuss the industrial and environmental significance of biosurfactants, emphasizing the potential of endosymbionts as sustainable sources of these amphiphilic compounds.