Asparaginase derived from Eschericia coli or Erwinia crysanthum is used in the treatment of juvenile Acute Lymphoblastic Leukemia (ALL). ALL cells cannot make asparagine due to the lack of L-asparagine synthetase gene. Asparaginase is therefore used as a chemotherapeutic treatment for ALL through hydrolysis of the L-asparagine to aspartic acid, which leads to starvation of the ALL tumor cells, then apoptosis. In addition, the asparaginase enzyme is used in the food industry. Asparaginase can be used to reduce acrylamide in baked food formed during the Milliard reaction. In this study, in this study, Rhodococcus rhodochrous DAP 96253 asparaginase enzyme activity is measured at varying temperatures (27 °C and 37 °C) and substrate concentrations, using colorimetric reagents, to determine the enzyme reaction stability with respect to these variables. Asparaginase enzyme activity increases by increasing the temperature to a specific optimal point, after that it declines. This accelerates the hydrolysis of L-asparagine to L-aspartic acid. The goal is to determine the optimum temperature for the enzyme function.