Myeloperoxidase (MPO) imaging is a non-invasive method to detect cells that produce the enzyme MPO that is most abundant in neutrophils, macrophages, and inflammatory monocytes. While lacking specificity for any of these three cell types, MPO imaging can provide guidance for further flow cytometry-based analysis of tissues where these cell types reside. Isolation of leukocytes from the intestinal tract is an error-prone procedure. Here, we describe a protocol for intestinal leukocyte isolation that works reliable in our hands and allows for flow cytometry-based analysis, in particular of neutrophils.