In the connective tissue, synovial fluid, and other tissues, hyaluronic acid (HA), plays a crucial role in the processes of inflammation, granulation tissue development, epithelial remodelling and the healing of chronic wounds in both mineralized and non-mineralized periodontal tissues. Therefore, this study aimed to investigate the effect of HA on PDL cell line proliferation in vitro at baseline, 3rd day, 5th day as well as to compare the rate of proliferation between the test and control group, baseline and day 3 as well as baseline and day 5 in the test group. Methods: A total of 30 samples of PDL fibroblast cell line were taken, which were divided into two groups consisting of 15 samples each. Group 1 includes the “control group” where the PDL fibroblast cell was unstimulated, while group 2 includes the “test group” consisting of stimulated PDL fibroblast cells by HA. Cell proliferation of the two groups was checked at baseline, 3rd and 5th day and the proliferation activity was determined by Alamar blue assay indicator. Results: Hyaluronic acid showed high PDL cell viability. It significantly increased cell proliferation compared to the controlled samples, and a significant increase in proliferation was seen between the 3rd and 5th day in the test group. Conclusion: HA sustained high PDL cell viability and promoted the growth of the cultured PDL fibroblast cell line. To better understand how HA affects periodontal growth, additional in vitro and animal study is required.