Different medicinal plants were screened for l-asparaginase. Asparagus racemosus exhibited highest activity (10.11 ± 0.12 U/ml) and specific activity (22.78 ± 0.28 U/mg of protein). A. racemosus was collected from Padmapuram garden of Araku valley, Visakhapatnam district. l-asparaginase activity was detected from the extracts of leaves and rhizome. Enzyme activity was maximum in the extract of rhizome (9.58 ± 0.12 U/ml) with specific activity (21.73 ± 0.28 U/mg of protein) compared with leaves which have the enzyme activity (1.85 ± 0.06 U/ml) with specific activity (2.68 ± 0.14 U/mg of protein). Optimum conditions for the extraction and assay of l-asparaginase from plant rhizomes were studied. Results showed maximum activity of l-asparaginase when the sample to buffer ratio was 3:6, sample weight (g)/buffer volume (ml) using 0.02 M of potassium phosphate buffer solution pH 8.2 at 40 °C. The l-asparaginase activity was 16.01 ± 0.06 U/ml with specific activity (26.81 ± 0.42 U/mg of protein) under optimum conditions.