The study was performed to explore physiological, non-enzymatic and enzymatic detoxification pathways of reactive oxygen species (ROS) in tolerance of Amaranthus tricolor under drought stress. The tolerant genotype VA13 exhibited lower reduction in growth, photosynthetic pigments, relative water content (RWC) and negligible increment in electrolyte leakage (EL), lower increment in proline, guaiacol peroxidase (GPOX) activity compared to sensitive genotype VA15. This genotype also had higher catalase (CAT), superoxide dismutase (SOD), remarkable and dramatic increment in ascorbate-glutathione content, ascorbate-glutathione redox and ascorbate-glutathione cycle enzymes activity compared to sensitive genotype VA15. The negligible increment of ascorbate-glutathione content, ascorbate-glutathione redox and ascorbate-glutathione cycle enzymes activities and dramatic increment in malondialdehyde (MDA), hydrogen peroxide (H2O2) and EL were observed in the sensitive genotype VA15. SOD contributed superoxide radical dismutation and CAT contributed H2O2 detoxification in both sensitive and tolerant varieties, however, these had a great contribution in the tolerant variety. Conversely, proline and GPOX accumulation were higher in the sensitive variety compared to the tolerant variety. Increase in ascorbate-glutathione cycle enzymes activities, CAT, ascorbate-glutathione content, SOD, and ascorbate-glutathione redox clearly evident that CAT, ascorbate-glutathione cycle and SOD played a significant activity in ROS detoxification of tolerant A. tricolor variety.