Background: The mode of harvesting autogenous bone grafts can affect the quality of harvested bone and viability. The present study was carried out to evaluate cellular viability, differentiation and proliferative power in autogenous bone graft harvested by auto chip maker versus milled bone block. Materials and Methods: 16 bone samples were harvested. In group I (n=8): auto chip maker (ACM) was used while in group II (n=8): bone blocks were harvested then milled. Flow cytometry, alkaline phosphatase activity, MTT assay, Alizarin Red Staining, and Real-Time PCR were performed Results: Group I had a significantly higher cell viability. MTT assay revealed a significant increase in cell proliferation in the ACM group. Both groups showed decrease in (RUNX2) gene expression over four days and increase in type 1 collagen alpha 1 and osteocalcin gene expression at 4th evaluation day with a statistical significant higher level of both markers in the ACM group. Conclusion: ACM group showed better osteoblasts viability, proliferation, differentiation, and calcified tissue formation.