L-Asparaginase, an enzyme drug used for the treatment of acute lymphoblastic leukemia and its effective usage in clinical arena is complicated owing to the significant Glutaminase side activity. To develop variants of the enzyme with reduced Glutaminase activity, in silico mutagenesis was done by replacing amino acids in the vicinity of the ligand binding site. It was identified that replacement of enzyme's active site amino acid Asp96 with Alanine decreased the Glutaminase activity by 30% and also increased the Asparaginase activity by 40%. Docking studies were carried out by Autodock 4.0 and binding energy for native enzyme when docked with glutamine was found to be −8.08 Kcal/mole, whereas for mutated protein it was found to be −5.97 Kcal/mole. It was also observed that replacement of active site with amino acids other than alanine did not show considerable change in both Asparaginase and Glutaminase activities. The designed enzyme model with reduced Glutaminase side activity may help to develop a variant of enzyme drug through protein engineering by site-directed mutagenesis and thus to produce a drug with reduced side effect for treating acute lymphoblastic leukemia in children.