Epithelial cell movement, key in development, tissue reparation, and tumor metastasis, is controlled by Rab GTPases and GEF cytohesins that regulate ARF GTPases and protein recycling. To visualize association of cytohesins with endosomal compartments, rapamycin is used to induce Rab-positive compartment migration to cell-center. This study optimized the rapamycin assay to quantify Rab-positive compartment interactions and cytohesin recruitment in HeLa cells. Future applications include a deeper understanding of protein interactions for application to drug discoveries.