Cancer stem cells are cancer cells that have the characteristics of healthy stem cells in the human body. The possibility of self-replication and differentiation in different cell types ensures their tumorigenicity while low proliferation rates with active membrane pumps and repair enzymes provide resistance to conventional chemotherapy. Their presence is proven in osteosarcoma and can be extracted from tumor mass based on the ability to grow in spheres - sarcospheres. Given that it is an extremely malignant tumor, the development of specific therapies targeting that population of cells is necessary to improve patient survival. Due to their resistance mechanisms, it is difficult to selectively target them. This study investigated the effect of ascorbic acid on osteosarcoma stem cells and its selectivity depending on the concentration of ascorbic acid and the number and origin of cells. The effects of ascorbic acid have been determined by viability tests, sarcosphere growth inhibition assays, flow cytometry, western blot and measurement of reactive oxygen species. In conclusion, ascorbic acid causes apoptosis and its lethal concentration is dependent on the number of osteosarcoma stem cells. Also, the level of GAPDH protein is undetectable, which suggests that ascorbic acid inhibits the pathway of glycolysis. The level of reactive oxygen species is increased in cells treated with ascorbic acid and it can be concluded that it acts as an oxidant.