Purpose We evaluated effects of asparaginase dosage, schedule, and formulation on CSF asparagine in children with acute lymphoblastic leukemia (ALL). Methods We evaluated CSF asparagine (2114 samples) and serum asparaginase (5007 samples) in 482 children with ALL treated on the Total XVI study (NCT00549848). Patients received one or two 3000 IU/m 2 IV pegaspargase doses during induction and were then randomized in continuation to receive 2500 IU/m 2 or 3500 IU/m 2 IV intermittently (four doses) on the low-risk (LR) or continuously (15 doses) on the standard/high risk (SHR) arms. A pharmacokinetic–pharmacodynamic model was used to estimate the duration of CSF asparagine depletion below 1 uM. Results During induction, CSF asparagine depletion after two doses of pegaspargase was twice as long as one dose (median 30.7 vs 15.3 days, p  < 0.001). During continuation, the higher dose increased the CSF asparagine depletion duration by only 9% on the LR and 1% in the SHR arm, consistent with the nonlinear pharmacokinetics of serum asparaginase. Pegaspargase caused a longer CSF asparagine depletion duration (1.3–5.3-fold) compared to those who were switched to erwinase ( p  < 0.001). The median (quartile range) serum asparaginase activity needed to maintain CSF asparagine below 1 µM was 0.44 (0.20, 0.99) IU/mL. Although rare, CNS relapse was higher with decreased CSF asparagine depletion ( p  = 0.0486); there was no association with relapse at any site ( p  = 0.3). Conclusions The number of pegaspargase doses has a stronger influence on CSF asparagine depletion than did dosage, pegaspargase depleted CSF asparagine longer than erwinase, and CSF asparagine depletion may prevent CNS relapses.