An increasing body of evidence indicates that a local islet immune response is not only limited to type 1 diabetes, but also is associated with islet dysfunction in type 2 diabetes. Recently, the presence of pancreatic CD68 macrophages within islet tissues was demonstrated by RT-PCR and immunohistochemical methods. However, the precise profile and activation status of intraislet leukocytes, which are present in both murine and human islets, are poorly defined. Here, we describe a detailed flow cytometry protocol designed to analyze both human and murine islets for intraislet leukocytes and leukocyte subsets. This approach permits the simultaneous identification of multiple intraislet leukocyte subsets, as well as their activation statuses. The use of flow cytometry-based approaches will advance the field of islet biology and help to identify unique changes in the immune cell composition that accompanies pathological islet inflammation and dysfunction in type 2 diabetes.