Objective: To investigate the population pharmacokinetics of delayed methotrexate (MTX) excretion in children with acute lymphoblastic leukemia (ALL). Materials and methods: A total of 1,659 plasma concentration samples of MTX from 190 patients with 1 - 4 courses (plasma concentrations > 0.1 mu mol/L) were collected in this study. The data analysis was performed using Phoenix NLME 1.3 software. The covariates included age, body surface area (BSA), body weight, alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine transaminase (ALT), total bilirubin (TBIL), and serum creatinine (SCr). The final model was validated by bootstrap resampling procedures (1,000 runs) and visual predictive check (VPC) method. Results: The data were best described by a two-compartment linear pharmacokinetic model. The mean values of clearance (CL) and distribution volume (V-d) of MTX were 6.53 L/h and 67.88 L, respectively. Analysis of covariates showed that BSA influenced the CL of MTX. Conclusion: The final model was demonstrated as appropriate and effective for assessing the pharmacokinetic parameters of delayed MTX excretion in children with ALL.