This article provides a comprehensive guide to the isolation, expansion, and differentiation of Adipose-derived Stem Cells (ASCs). ASCs are abundant in adipose tissue and exhibit multipotent properties, making them suitable for various regenerative medical applications. The authors detail the methods for isolating ASCs from human adipose tissue, which involves mincing the tissue, digesting it with collagenase, and centrifuging to separate the stromal vascular fraction (SVF) containing the ASCs. The SVF is then further processed to enrich the ASCs. The culture and expansion of ASCs are described, including the optimal conditions for proliferation and the steps to induce differentiation into adipocytes, osteocytes, chondrocytes, and neurons. The article also discusses the freezing and long-term storage of ASCs and provides protocols for assessing their differentiation potential. The authors conclude by highlighting the potential of ASCs in regenerative medicine and the need for further research to optimize their clinical application.This article provides a comprehensive guide to the isolation, expansion, and differentiation of Adipose-derived Stem Cells (ASCs). ASCs are abundant in adipose tissue and exhibit multipotent properties, making them suitable for various regenerative medical applications. The authors detail the methods for isolating ASCs from human adipose tissue, which involves mincing the tissue, digesting it with collagenase, and centrifuging to separate the stromal vascular fraction (SVF) containing the ASCs. The SVF is then further processed to enrich the ASCs. The culture and expansion of ASCs are described, including the optimal conditions for proliferation and the steps to induce differentiation into adipocytes, osteocytes, chondrocytes, and neurons. The article also discusses the freezing and long-term storage of ASCs and provides protocols for assessing their differentiation potential. The authors conclude by highlighting the potential of ASCs in regenerative medicine and the need for further research to optimize their clinical application.