Oxidative stress is well known to be involved in pathophysiology of several disorders. Valeriana wallichii (VW) root extracts is known for its expedient activities but its antioxidant and cytoprotective efficacy need to be explored further. In present study tertiary-butyl hydroperoxide (tert-BOOH) was used to induce oxidative stress in C6 glioma cells. Antioxidant activity of the VW root extracts were evaluated by chemical assays i.e. DPPH, ABTS and FRAP assay. Further effect of VW on tert-BOOH induced oxidative stress and mitochondrial damage was studied. Result of present study revealed that exposure of cells to tert-BOOH resulted in increase in cytotoxicity, reactive oxygen species (ROS) production and decrease in mitochondrial membrane potential, super oxide dismutase, catalase, glutathione peroxidase and glutathione reductase. On the other hand, pretreatment of cells with VW extracts ameliorated these damaging effects. Additionally, HPLC analysis revealed hesperidin as an active ingredient and concentration of heavy metal was found within the maximum permissible limits prescribed by WHO. In conclusion present study revealed the antioxidant and cytoprotective property of VW against oxidative stress.