In the intestinal epithelium, intraepithelial lymphocytes (IELs) coexist with intestinal epithelial cells (IECs). The IELs have an important role in defending the intestinal tract against pathogens and eliminating tumor cells. Anomalies in the absolute IEL count have been reported in various digestive diseases. IELs are typically counted using histologic techniques or under light microscopy after isolation of the epithelium. However, these techniques can introduce bias, which might account for the discrepancies in counts from one study to another. Here, we describe a flow cytometry assay for determining the absolute IEL count and the IEL/IEC ratio. We combined a conventional epithelial isolation method with a BD TruCount bead-based absolute counting technique to quantify IELs (CD45 CD326/EpCAM- CD103CD3) and IECs (CD45- CD326/EpCAM) in a C57BL/6 mouse model. Key features • Intraepithelial lymphocytes (IELs) play a crucial role in maintaining mucosal integrity and defending against pathogens. • Conventional manual counting of IELs using a hemocytometer relies heavily on the operator's expertise. • Flow cytometry offers a more standardized approach to cell counting. • Using TruCount beads to quantify IELs and intraepithelial cells (IECs) by flow cytometry and assess their ratio ensures reproducibility and comparison with immunohistochemical methods. Graphical Overview.