Adipose-derived mesenchymal stromal/stem cells (AD-MSCs) offer a promising cell source for regenerative medicine due to their easy accessibility and potential application across a wide range of disorders. This chapter describes a standardized enzymatic isolation protocol to extract AD-MSCs from both solid adipose tissue and lipoaspirate. Adipose tissue samples are initially washed with phosphate-buffered saline (PBS) to remove blood contaminants. Tissue is then digested enzymatically with type I collagenase under controlled temperature and agitation to release the stromal vascular fraction (SVF) containing AD-MSCs. The enzyme activity is neutralized using complete growth medium followed by centrifugation to separate the cell pellet from unwanted material. The resulting cell pellet is resuspended and filtered to eliminate residual tissue fragments. After seeding the cells in culture, AD-MSCs in the SVF are characterized by adherence to plastic and the expression of specific cell surface marker (e.g., CD73, CD90, CD105), confirming their identity. This protocol provides a method for isolating viable AD-MSCs for downstream applications.