Abstract: Background: Direct 3D printing of aligner trays involve printable materials; the study aims to investigate the in-vitro cytotoxicity of the direct-printed aligner using photopolymer resins and SmartTrackInvisalign tray for varying time intervals on 3T3 mice fibroblast cells using MTT assay. Materials and Methods: Directed printed aligner trays using two 3D printing materials with SmartTrackInvisalign tray were compared in this study. Samples were placed in Dulbecco’s Modified Eagle’s Medium (DMEM; 0.1 mg/mL) for 1,3,5& 7 days interval. Cell viability percentage was calculated, and data were analyzed using a one-way analysis of variance and post hoc tests (α = 0.05). Results: All materials exhibited slight cytotoxicity on MFCs with a visible trend of a significant increase in cell viability from day 1 to 7. Among the groups, the higher cytotoxicity was by E-Guard clear, and Dental LT, and the least cytotoxicity by Smartrack material. The highest level of cell viability and no cytotoxicity was exhibited by Invisalign (94.07% ± 3.00 of cell viability) at day 7. No statistically significant difference in viability percentage was seen between Dental LT and E-Guard material. Conclusions: SmartTrackInvisalign material (polyurethane) was found to be more biocompatible, followed by directly printed aligner materials (polymethylmethacrylate). Cytotoxicity was found to be more on the first day for all materials and gradually decreases as day’s progress. The results indicate the increased leaching of material during the initial period of use though the level of cytotoxicity is slight.