Innate lymphoid cells (ILCs) are present in most tissues within the body but have been most extensively studied within mucosal barriers such as the lung and intestine. Isolation of immune cells from such tissues requires enzymatic digestion, and the number and composition of the cells released are dependent upon robust protocols tailored to the tissue of study. Here, detailed methodologies to isolate ILCs from various barrier sites and their draining lymph nodes (LNs) are described. Flow cytometry staining and gating strategies for identification and quantification of ILCs are then provided. Combined, these provide an efficient means to study ILCs within the small intestine lamina propria, lung, ear skin, and LNs.