The three-dimensional culture models have been developed to promote better simulation of in vivo conditions of cellular interactions and phenotypic expressions. Since each cell line behaves differently and not all aggregate three-dimensionally, the objectives of this study were to standardize the conditions for cultivation spheroids produced from the human non-smal-cell lung cancer cell line NCI-H460, and evaluate the effects of cisplatin in this model. We investigated the best cell density for the formation of spheroids and their growth time in culture conditions. For this, spheroids were treated with different concentrations of cisplatin. The analysis was performed by measuring the diameter of the spheroids before treatment and measured 48 h after treatment and then every 3 days. The standard cell density conditions obtained was 10 x 104 cells. Spheroids were collected from the 7th day and isolated in a non-adherent matrix well (covered with 2% agar and culture medium), remaining viable until the 17th day. From the 8th day of isolated culture, the cisplatin promoted a reduction in the growth of spheroids dependent on the concentration used. Thus, it was concluded that it was possible to standardize the effective methodology for the formation and maintenance of spheroids from this cell line, as well as cisplatin inhibited the growth of the spheroids, suggesting its cytotoxic effect also cultures in three-dimensional model.