The defining morphological characteristic of endosomes is their multivesicular appearance, a product of the formation of intraluminal vesicles (ILVs) that bud from the limiting membrane and into the interior. It is poorly demonstrated how these ILVs arise, and what factors govern their formation and dynamics. In this study, we aimed to develop a robust methodology to monitor ILV formation and back-fusion by analyzing cargo known to traffic via ILVs in the endocytic system and to find novel candidates involved in this process. We have devised and tested a number of novel methods for monitoring the formation and dynamics of ILVs in the endocytic system and characterized the trafficking of ILV-dependent cargo.