Asparaginase (ASNase) is an important component of acute lymphoblastic leukemia (ALL) treatment, but is often discontinued because of toxicity. ASNase ( ) substitution was approved in 2011 for allergic reactions. has, however, been intermittently unavailable because of drug supply issues. The impact of substitution or complete ASNase discontinuation is unknown. Patients aged 1-30.99 years in frontline Children's Oncology Group trials for B-cell acute lymphoblastic leukemia between 2004 and 2011 (National Cancer Institute NCI standard risk SR: AALL0331; NCI high risk: AALL0232) were included. The number of prescribed pegaspargase (PEG-ASNase) doses varied by trial and strata. Maintenance therapy did not contain ASNase. Landmark analyses at maintenance compared disease-free survival (DFS) among those receiving all prescribed PEG-ASNase doses versus switching to but receiving all doses versus not receiving all ASNase doses. We included 5,195 AALL0331 and 3,001 AALL0232 patients. The cumulative incidence of PEG-ASNase discontinuation was 12.2% ± 4.6% in AALL0331 and 25.4% ± 0.8% in AALL0232. In multivariable analyses, NCI high-risk patients not receiving all prescribed ASNase doses had inferior DFS (hazard ratio HR, 1.5; 95% CI, 1.2 to 1.9; = .002) compared with those receiving all prescribed PEG-ASNase doses. Patients with substitution who completed subsequent courses were not at increased risk (HR, 1.1; 95% CI, 0.7 to 1.6; = .69). NCI SR patients who discontinued ASNase were not at elevated risk (HR, 1.2; 95% CI, 0.9 to 1.6; = .23), except when restricted to those with slow early response, who were prescribed more ASNase because of therapy intensification (HR, 1.7; 95% CI, 1.1 to 2.7; = .03). Discontinuation of ASNase doses is associated with inferior DFS in higher-risk patients. Our results illustrate the severe consequences of shortages.