L-asparaginase extracted from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukemia (ALL). The enzyme acts in the depletion of the amino acid L-asparagine from patients serum, which is essential for the neoplastic cell´s proliferation. However, this treatment with EcAII is hampered by human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB). Both hydrolases cleave EcAII, inactivating the enzyme and exposing epitopes associated with immune response. In this work we employed error-prone PCR to produce variants of EcAII with higher resistance to proteolytic cleavage by AEP and CTSB. A library of 1,128 clones was established and screened for asparaginase activity; among the mutants that maintained activity, one was resistant to CTSB proteolytic cleavage and two were resistant to both CTSB and AEP. The effect of each mutation in EcAII quaternary structure affecting the AEP and CTSB proteolytic recognition sites was explained using Molecular Dynamics Simulation. The mutant EcAII with higher resistance to CTSB could allow a prolonged half-life in human serum diminishing dose and frequency of protein administration, decreasing immune response.