Enteric glia, integral to the enteric nervous system (ENS), are crucial for intestinal motility, secretomotor function, and immunity. These glia occupy diverse niches from the serosa to the lumen, yet their transcriptional diversity across these compartments remains incompletely understood. Traditionally, studies on enteric glia have mostly focused on the myenteric plexus, omitting the large populations of glia located in the circular muscle and mucosa. Here, we present a method to rapidly isolate enteric glia from the muscularis externa and mucosal compartments of the mouse intestine in tandem, allowing direct comparative and compartment-specific analyses of each population. This protocol circumvents the need for traditional and labor-intensive longitudinal muscle myenteric plexus (LMMP) peeled tissue preparations by enabling rapid cell isolation suitable for many downstream applications, ranging from transcriptional profiling to cell culture. We detail a protocol for mechanically separating adult small intestinal layers, dissociating cells from the mucosal and muscularis compartments, and sorting these cells by flow cytometry. This method is applicable to both small and large intestines and has been validated in mice from weaning to adulthood. The ability to isolate distinct populations of enteric glia will enable functional interrogation of their niche-specific roles, advancing our understanding of their diverse contributions to ENS biology and pathology.