Cyclodextrins are cyclic molecules with a hydrophilic external structure and a hydrophobic internal cavity. Due to their chemical structure, cyclodextrins can form complexes with hydrophobic or amphiphilic molecules, including lipids. This property has resulted in the widespread use of cyclodextrins for characterization and manipulation of lipids in biological membranes. In this chapter, the use of cyclodextrins for characterization of lipids in the cell plasma membrane is discussed. First, the chemical structure of different cyclodextrins is explained. Then, the current knowledge on the mechanisms of cyclodextrin–lipid interaction and the role of cyclodextrin and lipid chemical structure in the outcome of such interactions is summarized. The use of cyclodextrins for manipulation of plasma membrane lipids and its application to study membrane lipid rafts is also reviewed. Finally, future directions in the field and current opportunities to utilize cyclodextrins to provide a better understanding of the composition and organization of lipids in the plasma membrane are discussed.