Introduction: The demand for aesthetic orthodontic appliances, namely clear aligners, has increased in recent years. However, the cytotoxic potential of these materials needs to be evaluated to ensure their safe use. This work intends to evaluate the in vitro cytotoxicity of Invisalign® and Spark™ aligners in 3T3-L1, MRC-5 and Vero cell lines.Materials and Methods: Conditioned media by the refereed aligners were prepared and cells were exposed for 24 and 48 hours. The metabolic activity was determined through the MTT assay, the cell viability, types of death and changes in the cell cycle by flow cytometry and the cell morphology evaluated by staining with May-Grünwald Giemsa.Results: Considering the Invisalign® conditioned medium, the Vero cell line revealed a significant decrease in metabolic activity at all concentrations at 24 hours of exposure and at 100%, 50%, 25% and 12.5% concentrations after 48 hours of incubation. Comparing the two aligners, Invisalign® caused a greater reduction in metabolic activity than Spark™. The Vero cell line, when exposed to the Invisalign® conditioned medium also shows a decrease in the percentage of living cells and an increase in cells undergoing apoptosis and late apoptosis/necrosis. When comparing the two aligners, there is a significant increase in cells in late apoptosis/necrosis in the Invisalign® group. The MRC-5 cell line, when exposed to the Invisalign® conditioned medium, shows a decrease in the percentage of living cells and an increase in cells in apoptosis. There were no statistically significant differences in the cell cycle evaluation of 3T3-L1 and Vero cells. The evaluation of cell morphology in these two lines revealed morphological characteristics of apoptosis and necrosis.Discussion: The cytotoxic effect depends on the cell line and the type of material. The Invisalign® aligner caused significant reductions in metabolic activity and cell viability, while SparkTM caused a significant reduction only in metabolic activity. The results obtained support that the aligners´ components are released into the medium and are responsible for the observed biological effect. Differences in the constituent materials of the two brands may account for the increased cytotoxicity of Invisalign®.Conclusion: Both brands have biological effects on the studied cell lines, however, the Invisalign® aligner has greater cytototoxicity than SparkTM. More studies are needed to investigate possible local and systemic effects, to ensure safety in the clinical use of these devices.