Rhodiola imbricata (Roseroot) is a perennial plant distributed in South Asia viz., Pakistan, Nepal, China and India. In India it is endemic to Trans Himalayan cold desert of Ladakh. It is a well known medicinal plant in the Amchi system of medicine (Tibetan system of medicine) being used in various ailments like anti-stress, radio-protective, anticancer, anti-inflammatory agent, adaptogen etc. The aim of this study was to assess the in vitro potential of methanol extract of Rhodiola imbricata roots. The DPPH activity of the extract (0.1–1.2 mg/ml) was increased in a dose dependent manner, which was found in the range of (39.55-70.76%) as compared to ascorbic acid (46.78 –81.47%). The IC50 values of methanol extract in DPPH radical, nitric oxide, hydroxyl radical were obtained to be 0.33, 0.47, 0.58 mg/ml, respectively. However, the IC50 values for the standard ascorbic acid were noted to be 0.42, 0.43, 0.51 mg/ml, respectively. Measurement of total phenolic content of the methanol extract of R. imbricata was achieved using Folin–Ciocalteau reagent containing 185.7 mg/g of phenolic content, which was found significantly higher when compared to reference standard gallic acid. The results obtained in this study clearly indicate that R. imbricata has a significant potential to use as a natural anti-oxidant agent.