Abstract: Serum-free cell culture methods are now routinely support mammalian cell growth, a practice adopted for ethical, scientific and safety concerns. The HeLa-S3 cell line is a subclone of the HeLa cell line that can grow in Serum-Free Medium (SFM) as well as suspension cultures. In order to optimize its culturing conditions in SFM, the present study investigated the efficacy of insulin and L-glutamine additives as biotic factors as well as osmotic stress as abiotic factor, all affecting growth kinetics and metabolism. Insulin was used with different concentrations ranging from 10 to 50 mg L-1. It was found that cell growth is dependent on insulin up to a concentration of 25 mg L-1 at which maximum cell number as well as cell viability were achieved. Similarly, L-glutamine was used in the range of 3 to 8 mmol L-1 and was found optimum at 3 mmol L-1. However, osmotic stress using saline solution addition showed that osmolality in the range of 314 to 350 mOsm kg-1 is preferable to cells. The study also showed the successful sequential cell adaptation from adherent culture mode to suspension culture in which cells were able to grow in small clumps of spherical-shaped cells. Based on this cultivation strategy, HeLa-S3 cells were completely adapted to proliferate suspended in serum-free medium with sustained growth kinetics and physiological properties.