Gut microbiome is an essential partner in the maintenance of gastrointestinal (GI) homeostasis, through its interaction with both epithelium, immune cells, and enteric nervous system (ENS). Under pathological conditions, disturbances of resident bacteria composition and/or metabolism frequently combine with ENS dysfunction. Enteric glial cells (EGCs), accompanying neurons across all the layers of the GI tract, can orchestrate neuronal activity and immune response to microenvironment perturbation, but their interaction with the components of microbiota has been poorly investigated. Indeed, although a reciprocal influence between gut microflora and EGC has been suggested, further research is needed to elucidate the mechanisms governing this crosstalk and its implications in health and disease. To this end, in vitro studies appear necessary as they allow for dissection and selective analysis of microbiota-to-EGCs signaling without the intestinal contour. This chapter has the objective to describe a method to process fecal material from either animals or humans for in vitro studying microbiota-mediated modulation of EGCs activity. It also illustrates the advantage of using whole fecal supernatant (FS) over selected supernatant and/or living bacteria to in vitro mimic microbiota signaling and introduce the possibility to treat EGCs with the conditioned medium of FS pre-treated epithelial cells.