The ESCRT machinery (endosomal sorting complex required for transport) is an evolutionarily highly conserved multiprotein complex involved in numerous cellular processes like endocytosis, membrane repair, or endosomal long-distance transport. In fungal hyphae, endocytosis and long-distance mRNA transport are tightly linked, as endocytotic vesicles are also the key carrier vehicles for mRNAs. Studying the regulatory component Did2 (CHMP1) in the plant pathogen Ustilago maydis revealed that loss of Did2 resulted in disturbed endosomal maturation, thereby causing defects in microtubule-dependent transport of early endosomes. Here, we describe methods and protocols that allow studying the role of ESCRT components during endosomal transport. We present experimental strategies to analyze U. maydis ESCRT mutant phenotypes and test complementation with heterologous components, such as ESCRT regulators from Drosophila melanogaster.