Background: Extract from the roots of Ashwagandha plant has been used through generations for its positive effect as an anti-stress, anti-tumor, anti-arthritic, and neuro-regenerative agent. The compounds have shown considerable effect as a thyroid disrupting agent also. Extremely contradictory findings have been shown in both animal and human studies regarding the effect of Ashwagandha on thyroid levels, and hence it is important to monitor the safety and efficacy in pre- clinical models before we shift to clinical trials for the same. Objective: To evaluate the thyroid disrupting potential of the Ashwagandha root extract (ARE) on Zebra Fish (Danio rerio) model. Methodology: A total of 105 (7 groups of 15 each) transgenic Zebra fish embryos (tg: mCherry) at 2 days post-fertilization stage were included to study the effect of ARE on the developing thyroid gland. Potassium perchlorate (KOCl), a known thyrotoxic substance was used as a positive control and dimethyl sulfoxide (DMSO) was the vehicle control. The effect on thyroid glands was assessed using a thyroglobulin reporter gene fluorescence assay. Five serial concentrations from 1 mcg/ml to 50 mcg/ml of ARE were used. On day 5 post-fertilization, images were analyzed under a fluorescence microscope for intensity variations. Result: The result from this study showed no differences in the expression of thyroglobulin reporter gene with Ashwagandha root extract. Conclusion: The Ashwagandha root extract did not show any effect on thyroid gland in developing zebra fish embryos and can be considered safe with respect to its effects on thyroid gland.