L-asparaginase is an effective anti-tumor agent for acute lymphoblastic leukemia. This work presents the development of an activity determination of L-ASNase preparations for pharmaceutical quality control purposes, in accordance with analytical Quality by Design principles. Critical method attributes, the absorbance at 450 nm (A450) of the Nessler product as well as its variability, were evaluated as a function of critical method variables, by using experimental designs. The design space of the enzyme activity assay was defined (Nessler method: C(KI)/C(HgI2) of 1.90-1.95, C(NaOH)/C(HgI2) of 17.0-18.0, C(HgI2final) of 20-40 mM and time of 10-40 min; enzyme activity conditions: temperature range of 36.6-37.4 °C, pH range of the KH2PO4 buffer from 7.1 to 7.7, KH2PO4 buffer concentration: 0.18-0.22 M and L-Asn concentration of 18-22 mM), leading to a final enzyme activity assay method. A control strategy was ultimately implemented using system suitability tests.