The intestinal lamina propria (LP) is a leukocyte-rich cornerstone of the immune system owing to its vital role in immune surveillance and barrier defense against external pathogens. Here, we present a protocol for isolating and analyzing immune cell subsets from the mouse intestinal LP for further downstream applications. Starting from tissue collection and cleaning, epithelium removal, and enzymatic digestion to collection of single cells, we explain each step in detail to maximize the yield of immune cells from the intestinal LP. Display omitted •Protocol for the isolation of murine leukocytes from the intestinal lamina propria•Comparative analysis of the isolation efficiency of three widely used digestion enzymes•Assessment of intestinal immune cells using minimal 8-color flow cytometry panels•Advice for choosing the digestion enzyme based on the cell of interest to maximize yield Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The intestinal lamina propria (LP) is a leukocyte-rich cornerstone of the immune system owing to its vital role in immune surveillance and barrier defense against external pathogens. Here, we present a protocol for isolating and analyzing immune cell subsets from the mouse intestinal LP for further downstream applications. Starting from tissue collection and cleaning, epithelium removal, and enzymatic digestion to collection of single cells, we explain each step in detail to maximize the yield of immune cells from the intestinal LP.