Drosophila intestinal tumors show an extended cellular heterogeneity. We devise a protocol to assess tumor cell heterogeneity by employing nuclear size measurement and immunofluorescence-based cell lineage analysis. We describe steps for intestinal dissection, staining, and imaging, followed by detailed procedures for nuclear size analysis. This approach detects overall heterogeneity across the entire tumor cell population and deviations within specific cell populations. The procedure is also applicable for analyzing the heterogeneity of wild-type intestinal cells in various contexts. For complete details on the use and execution of this protocol, please refer to Pranoto et al..