Adipose-derived mesenchymal stromal cells are promising as a regenerative therapy tool for defective tissues in mesenchymal lineage, including fat, bone, cartilage, and blood vessels. In potential future clinical applications, adipose-derived stem cell cryopreservation is an essential fundamental technology. The aim of this study is to define an adequate protocol for the cryopreservation of adipose-derived mesenchymal stromal cells, by comparing various protocols so as to determine the effects of cryopreservation on viability and chondrogenic differentiation potential of adipose-derived stem cells upon freeze-thawing of AT-MSCs colonies cryopreserved with standard and modified protocols, using flow cytometry and confocal microscopy. The study concludes that adipose-derived mesenchymal stromal cells could be long-term cryopreserved without any loss of their proliferative or differentiation potential.