GM-CSF is a crucial component in immunological and inflammatory responses. Recombinant human GM-CSF (rhGM-CSF), generated using recombinant DNA technology, offers important therapeutic and research opportunities. The objective of this study was to isolate and analyse rhGM-CSF, assess its biological functionality, and determine the most favourable experimental parameters for conducting studies. The methods employed encompassed the optimization of cell yield, measurement of proteins, bioassays to evaluate cell proliferation triggered by GM-CSF, and cytokine production. An equilibrium between the frequency of occurrence and the ability of the test to detect it.Protein quantification and trypan blue exclusion assay confirmed high purity, and optimal abundance was detected at 0.4 units/ml GM-CSF. Dose-response assays in TF-1 cells identified 1.7 ng/mL as optimal for proliferation, whereas potency assays and dot blot analyzes reconfirmed the protein identified at 800 IU/mL peak concentration by the WST-8 assay emphasize purity and integrity. Quantitative ELISA revealed reliable levels of GM-CSF, while significant induction of IL-1β in THP-1 cells revealed its inflammatory effect. This study clarifies the biological activity of rhGM-CSF and highlights the importance of immune modulation in clinical and research settings. The findings lay a strong foundation for future research and development in biopharmaceutical and clinical domains.