Adipose-derived stem cells (ASCs) reside in the stromal compartment of adipose tissue and can be easily harvested in large quantities through a clinically safe liposuction procedure. ASCs do not induce immunogenic reactions and rather exert immunosuppressive effects. Therefore, they can be used for both autologous and allogeneic transplantations. They hold great promise for cell-based therapies and tissue engineering. A prerequisite to the realization of this promise is the development of successful cryopreservation methods for ASCs. In this chapter, we describe a xeno-free- and chemically defined cryopreservation protocol, which can be used for various clinical applications of ASCs.