Sphingolipids (SLs) are concentrated at the plasma membrane where they play important roles in cell-cell communication, host-pathogen interactions, and cell signaling events. Our laboratory has used fluorescent SL analogs and SL-binding toxins to elucidate mechanisms by which SLs are internalized by endocytosis and subsequently sorted and transported to various intracellular compartments. These studies have relied on the use of temperature shift protocols, co-localization studies with compartment-specific markers, selective biochemical treatments that inhibit specific endocytic mechanisms, and the expression of dominant negative proteins (e.g., rabs) that block specific steps in transport. These methods are presented here so that they can be utilized by others for the study of endocytic trafficking of lipids and other molecules.