Abstract: The seeding density of cells, which directly influences the resultant adherent cell density, is a crucial factor affecting the accuracy of subsequent biological experiments. In this study, Hela cell line was used as a model, with two commonly used seeding densities in in vitro experiments: 3000 cells/well and 4000 cells/well. Differences in post-seeding coverage area, growth rates, and the outcomes of drug experiments with traditional chemotherapy and novel targeted therapeutics were compared between these two seeding densities. Significant statistical differences were observed in coverage area between the two groups (P < 0.05). Furthermore, the growth rate at 4000 cells/well was found to be higher than at 3000 cells/well. During the use of the conventional chemotherapy drug, Cisplatin, there was no significant difference in drug inhibition rates between the two cell densities at days 1, 3, and 5. However, when the cell cycle inhibitor Palbociclib was used, significant differences in drug inhibition rates were observed on days 1, 3, and 5. Therefore, the initial seeding density of cells can significantly impact the evaluation of some specific drugs.