Osteogenesis, which refers to the formation of bone tissue, can be studied in vitro to elucidate the mechanisms of bone development, identify key factors and cytokines, and enhance potential regenerative medicine applications in tissue engineering and transplantation. Adipose-derived stromal/stem cells (ASCs) are particularly promising for these studies due to their capacity to differentiate into various mesodermal lineage cells, including osteoblasts. To induce osteogenic differentiationOsteogenic differentiation, ASCs are cultured in a complete growth medium (CGM) supplemented with β-glycerophosphate, ascorbate-2-phosphate and dexamethasone over a period of at least 3 weeks. Osteogenic differentiation can be assessed through the deposition of hydroxyapatite, a key component of mineralized bone matrix, and the activity of alkaline phosphatase, an enzyme crucial for matrix calcification. This study provides a protocol for differentiating ASCs into osteoblasts using osteogenic differentiationOsteogenic differentiation medium (ODM) and evaluating the differentiation using Alizarin Red S (ARS) staining and alkaline phosphatase (ALP) assays. The methodology includes the preparation of CGM and ODM, as well as the specific procedures for ARS staining and ALP activity assays. The ARS staining involves fixation of cells, staining, and quantification of hydroxyapatite deposition, while the ALP assay measures enzyme activity. Both assays are normalized to cell counts, determined through DAPI staining and microscopic analysis. The results of these assays are intended to provide an accurate evaluation of osteogenic differentiationOsteogenic differentiation efficiency in ASCs.