In view of the constant increase of nanotechnology and nanomaterials applications in our daily life, to determine whether they are safe, “in vitro” and “in vivo” screening methods are needed. Obviously, application of models that are similar to the physiological tissues process of the human body could be a better candidate. The three-dimensional spheroid method, spheroid were generated using commercial microplates, has many benefits (in comparison with traditional methods or monolayer cell culture) such as the growth of the cells in 3D, similar to the body's physiological tissue, an alternative for animal models, cell-to-cell interactions, and better cell signaling. In this study, the toxicity of silver nanoparticles by using three factors such as metabolic activity, live/dead assay, and spheroid surface area was evaluated using two different methods (2D vs 3D) under treatment with various concentrations of silver nanoparticles at different times. The results showed that different cells types, cancer and/or normal lung cells, have significant differences. In addition, it was observed that distinct differences in terms of cytotoxicity of silver nanoparticles between 2D and 3D culture systems and also the rate of growth/non-growth of spheroids are highly depended on cell type and various concentrations have fundamental importance in such studies. The present study provides evidence that cellular dimensions (3D vs 2D) play a pivotal role in the results and outcomes of inflammation and cytotoxicity with nanoparticles due to the spatial-temporal structure.