Background: Adipose tissue (AT) is a rich source of mesenchymal stem cells (MSCs), however, there is no standardized protocol for stem cell isolation and culture. This leads to inconsistency of the results and limits the comparison of the data from different laboratories. Our aim was to provide an applied protocol for ASCS isolation and expansion, study the cell behavior and define their cellular surface markers. ASCs were cultured from both resected adipose tissue (RAT) obtained following abdominoplasty or breast reduction and lipoaspirates (LPA) following laser-free liposuction. Method: the protocol entailed coculturing of stromal vascular fraction (SVF) with RAT as raw pieces using DMEM medium with varying glucose concentration. The coculture protocol aimed to mimic the normal physiological conditions required for cell growth. ASCs were immunophenotyped to define their MSCs surface markers by flowcytometry. Results: ASCs were isolated from coculturing RAT with SVF with fibroblast-like adherent cells morphology. The ASCs yield isolated from LPA was significantly greater than from RAT on day 14 and 28 (p = 0.002, <0.001, respectively). Significant increase in ASCs proliferation rate was detected when ASCs were cultured under high glucose (4.5 g/L) compared to low glucose (1 g/ L) condition on day 7 and 14 (p = 0.04, 0.015, respectively). ASCs isolated from both protocols were positive for CD34, CD49d, CD73, CD90 and CD105 and negative for CD3, CD14, CD19, CD45 and HLA-DR. Conclusion: We concluded that the cells harvested by our protocol were ASCs. Hence, our method can be an efficient isolation tool to obtain primary ASCs under culture conditions mimicking normal physiological status. This will help in providing ASCs which can be similar to cells in human tissue for further study.