Abstract: Different parts of plant species belonging to Solanaceae and Fabaceae families were screened for L‐asparaginase enzyme (E.C.3.5.1.1.). Among 34 plant species screened for L‐asparaginase enzyme, Withania somnifera L. was identified as a potential source of the enzyme on the basis of high specific activity of the enzyme. The enzyme was purified and characterized from W. somnifera, a popular medicinal plant in South East Asia and Southern Europe. Purification was carried out by a combination of protein precipitation with ammonium sulfate as well as Sephadex‐gel filtration. The purified enzyme is a homodimer, with a molecular mass of 72 ± 0.5 kDa as estimated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresisand size exclusion chromatography. The enzyme has a pH optimum of 8.5 and an optimum temperature of 37 °C. The Km value for the enzyme is 6.1 × 10−2 mmol/L. This is the first report for L‐asparaginase from W. somnifera, a traditionally used Indian medicinal plant.