The mechanism for the movement of cholesterol from intracellular sites to their ultimate cellular destination is an unresolved question of fundamental importance to cell biology and medicine. Thus, defining mechanisms of intracellular cholesterol transport and identifying the cellular factors involved are therefore of great interest. Cayman’s Cholesterol Cell-Based Detection Assay includes filipin III, fixative, and wash buffer in a ready to use format. It provides a simple fluorometric method to study mechanisms and biological factors that regulate cholesterol metabolism or movement within cells. A cholesterol trafficking inhibitor, U-18666A, is included as a positive control.