Calcium (Ca2+) imaging is a versatile technique to study cellular activity and is a particularly important tool to study electrically passive cells such as glia. Enteric glia play important roles in gut physiology, and their activity can be captured using Ca2+ imaging. Ca2+-encoded activities command glial processes such as gliotransmission and intercellular signaling. This chapter describes a protocol that will allow users to study glial Ca2+ activity in ex vivo whole-mounts of circular muscle-myenteric plexus. This chapter will include comprehensive steps to execute Ca2+ imaging experiments, data extraction, and analysis.