Background: Euphorbia neriifolia leaves belonging to family euphorbiaceae being used traditionally for the treatment of asthma, bronchitis, tumours, leucoderma, piles, inflammations and ulcers. Earlier studies reported various chemical constituents such as euphorbon, resin, euphorbol and euphol. Hypothesis of work: On the basis of recent studies, it is concluded that reactive oxygen species are involved in several diseases. Hence the present study investigated to isolate the compounds from leaves of E. neriifolia and perform its antioxidant activity. Methods: Column chromatography and paper chromatography was used to isolate the pure constituent from the extract and antioxidant activity was performed using free radical scavenging and inhibition of lipid peroxidation assay. Results: Extractive value of ethanolic extract obtained was 36.52%w/w,which upon partitioned with ethyl acetate to get ethyl acetate fraction was 50.00%w/w. Column chromatography of ethyl acetate fraction (26-34) yielded cycloartenol upon repeated preparative TLC. The phytochemical screening of ethanolic extract of E. neriifolia revealed the presence of sterols, glycosides, phenolic compounds, flavonoids, saponins, carbohydrates and proteins and amino acids. The ethyl acetate fraction showed positive test for presence of glycosides, steroids, triterpenoids and flavonoids. The antioxidant activity of isolated cycloartenol was determined by measuring its 1, 1 diphenyl-2-pycryl hydrazyl (DPPH) radical scavenging activity and ability to inhibit lipid peroxidation. Cycloartenol exhibited strong antioxidant activity. Concentrations of 10, 20, 40, 60, 80, and 100μg /mL showed 34.56, 39.78, 44.68, 61.98, 66.46 and 72.87% inhibition of lipid peroxidtion respectively. In addition cycloartenol had effective DPPH free radical scavenging activity at 40μg/mL. Conclusion: It is concluded from present study that cycloartenol isolated from E. neriifolia leaves exhibited significant antioxidant activity and is a source of natural antioxidants that may be used in prevention of various diseases.