In immunological terms, the mammalian digestive tract, ranging from oral mucosal surfaces through stomach, small intestine and ending in the colon, comprise tissue compartments that are as diverse as the microbial communities inhabiting them. The essential contribution of the microbiota to food processing and nutrient supply has been described and accepted long time ago. What was discovered much more recently is the importance of the microbiota for the development and shaping of host immune responses. Thereby, the presence of commensals secures not only the immune homeostasis within the digestive compartment but grants proper immune responses throughout the mammalian body, including immunologically privileged sites like the CNS. One of the most prominent examples of recent research efforts was the investigation of germ-free laboratory animals, which present with a generally underdeveloped immune response but at the same time are also less prone to develop certain autoimmune conditions. Although the research on microbiota-immune interactions has grown exponentially in the past two decades, we are still only in the beginning to understand the complex interaction and interdependence of our immune system with these commensals. The aim of this Methods Collection is to compile state-of-the-art protocols for the investigation of microbiota immune interactions, including but not limited to in vivo test and imaging procedures, cell and tissue isolation protocols, sequencing approaches as well as cellular ex vivo and in vivo analyses. Together, these methods should help to standardize and fuel research on this emerging topic.