Jatropha curcas L. is a soft-wooded shrub found both in wild and cultivated conditions across the country. The seeds of J. curcas have been used as a purgative, antihelminthic, abortifacient as well as for treating ascites, gout, paralysis, and skin diseases. Present study includes physicochemical, phytochemical, antioxidant and HPTLC analysis of J. curcas seeds. Ethanolic, aqueous and hydroalcoholic extracts from J. curcas were screened for antioxidant evaluation using DPPH free radical scavenging activity. Protocatechuic and Gallic acid, two potential antioxidants present in this species, has been studied through HPTLC which may be utilized for the proper standardization of the drug. In J. curcas, IC50 of ethanolic, aqueous and hydroalcoholic extract were found to be 46 ± 3.46 μg/ml, 36.66 ± 0.57 μg/ml and 32.66 ± 1 μg/ml respectively. Ascorbic acid is used as standard which showed IC50 of 2.66 ± 0.11 μg/ml. HPTLC studies showed the presence of protocatechuic acid and gallic acid at Rf 0.69 and 0.32 with r2 of 0.998 and 0.994 respectively with mobile phase toluene: ethyl acetate : formic acid (5:5:0.5). J. curcas seed extract showed significant in vitro antioxidant activity, 50% hydroalcoholic extract showed the most potent activity. Quantification of protocatechuic and Gallic acid in 50% hydroalcoholic extract of J. curcas has been performed and was found to be 0.146% and 0.092% respectively.