Human granulocyte colony stimulating factor (hG-CSF) is one of a family hematopoietic growth which stimulates the proliferation and differentiation of neutrophil precursor cells and enhances some of the functional properties of mature neutrophils. hG-CSF has been well known to widely use to treat different forms of neutropenia, especially neutropenia caused by chemotherapy. Currently, some reports show that an increasing of neutropenia patient impulses the development of production of G-CSF. Among the cell lines used for biological assay of G-CSF, MNFS-60 has made it possible to estimate levels of G-CSF with great ease. This cell line has been used to determine bioactivity of commercial G-CSF (Neupogen-Amgen) which is approved by US Food and Drug Administration (FDA). Until now, there is no report on validation of biological assay of G-CSF using M-NFS-60 cell line. Especially, in Viet Nam, there is no study on validation of biological assay of cytokine as well. In this study, the authors investigated the bioactivity of G-CSF samples in MNFS-60 cell line by using proliferation assay. The authors studied some parameters as the concentration of hG-CSF, cell density, and time of hG-CSF treatment to optimize these conditions. the results showed that the optimal concentration of hG-CSF uses for this bioassay is from 10-2 to 106pg/ml, the cell density is 105cells/ml and the time to treat with hG-CSF is 48 hours. This process was efficient and used to determine the recombinant hG-CSF's activity, including samples before and after purification as well as lyophilization. The unit activities of these samples determined by using the process showed that these samples have the same effect on M-NFS-60 cell line comparing to commercial sample (Neupogen).