Adipose-derived stem cells (ASCs) are multipotent cells isolated from adipose tissue, offering potential for regenerative medicine. This article details methods for isolating, expanding, and differentiating ASCs. ASCs are isolated from adipose tissue through enzymatic digestion and centrifugation, followed by plating and expansion in culture. The cells can be differentiated into adipogenic, osteogenic, chondrogenic, and neural lineages using specific growth factors and media. Adipogenic differentiation involves inducing cells to accumulate lipids, while osteogenic differentiation leads to mineralization. Chondrogenic differentiation is achieved through pellet culture, and neural differentiation is done by forming neurospheres. ASCs have significant therapeutic potential due to their ability to differentiate into multiple cell types and their accessibility from adipose tissue. The protocols described can be adapted for different adipose tissue sample sizes. Challenges remain in clinical application, including the need for large-scale manufacturing and quality control. This review provides detailed protocols for ASC isolation, expansion, and differentiation, highlighting their importance in regenerative medicine and drug discovery.Adipose-derived stem cells (ASCs) are multipotent cells isolated from adipose tissue, offering potential for regenerative medicine. This article details methods for isolating, expanding, and differentiating ASCs. ASCs are isolated from adipose tissue through enzymatic digestion and centrifugation, followed by plating and expansion in culture. The cells can be differentiated into adipogenic, osteogenic, chondrogenic, and neural lineages using specific growth factors and media. Adipogenic differentiation involves inducing cells to accumulate lipids, while osteogenic differentiation leads to mineralization. Chondrogenic differentiation is achieved through pellet culture, and neural differentiation is done by forming neurospheres. ASCs have significant therapeutic potential due to their ability to differentiate into multiple cell types and their accessibility from adipose tissue. The protocols described can be adapted for different adipose tissue sample sizes. Challenges remain in clinical application, including the need for large-scale manufacturing and quality control. This review provides detailed protocols for ASC isolation, expansion, and differentiation, highlighting their importance in regenerative medicine and drug discovery.