L-asparaginase (ASNase) is a standard agent in the treatment of acute lymphocytic leukemia, but its mechanism of elimination has not yet been determined. Possible methods include hepatic and renal clearance, although renal clearance has been deemed unlikely. A physiologically-based pharmacokinetic model, including plasma, liver, kidney, muscle and gastrointestinal tract compartments, was constructed for ASNase to study the factors that affect its elimination, and to determine if renal clearance can be ruled out. Simulations were performed for hepatic and renal clearances alone and hepatic and renal clearances together. The model was fit to animal data to estimate the tissue-to-plasma partition coefficients and clearance rates. The optimal partition coefficients were 0.01-0.077 for the liver, kidneys and gastrointestinal tract, and 0.001-0.0077 for the muscles. Based on the model, the following methods of ASNase elimination are possible: (1) hepatic only, (2) hepatic and renal, (3) renal only and (4) other methods not considered here.