Mesenchymal stem cells (MSCs) are multipotent progenitor cells that can differentiate into various cell types, including adipocytes, osteocytes, and chondrocytes. Due to their regenerative potential, MSCs have gained significant interest in both clinical and research applications, particularly in tissue repair, immune modulation, and cell-based therapies. This protocol outlines a standardized method for isolating MSCs from human adipose tissue obtained through liposuction. The process involves careful preparation of reagents, collection and processing of fat, and enzymatic digestion with collagenase to dissociate the adipose tissue. After digestion, the MSCs are separated from other cellular components through centrifugation, lysis, and culture in a suitable medium. The protocol ensures a high yield and viability of MSCs for subsequent culture and expansion. This approach can be adapted for clinical or experimental studies requiring autologous or allogeneic MSCs derived from human adipose tissue.