This unit describes a cryopreservation procedure using an enzyme-free dissociation method to harvest cells and preserve cells in albumin-free chemically defined E8 medium for human pluripotent stem cells (hPSCs). The dissociation by EDTA/PBS produces small cell aggregates that allow high survival efficiency in passaging and cryopreservation. Cryopreservation in E8 medium eliminates serum and other animal products, and is suitable for dealing with the increasing demand for high-quality hPSCs in translational research. In combination with the special feature of EDTA/PBS dissociation, the protocols in this unit allow for efficient cryopreservation in a more time-saving manner.