Title: Assessing the Risk: Predictive Value of Asparaginase Assay Levels for Calaspargase Induced Hypersensitivity in Pediatric Patients Background: Calaspargase pegol-mknl, the only pegylated asparaginase available for pediatric acute lymphoblastic leukemia (ALL) patients in the U.S., is associated with hypersensitivity and neutralizing antibody development, which can reduce therapeutic efficacy. This study evaluates whether serum asparaginase activity (SAA) levels can predict hypersensitivity and antibody formation in pediatric ALL patients . Methods: A retrospective chart review was conducted at Cook Children’s Health Care System (December 2022–November 2024) of patients aged 0–21.5 years treated with calaspargase who experienced hypersensitivity. A control group of patients without hypersensitivity or neutralizing antibodies was used for comparison. Data included demographics, SAA levels, and dosing details. Descriptive statistics summarized outcomes, and Fisher’s exact and chi-square tests assessed associations between patient-specific factors and hypersensitivity (p<0.05). Results: Of 121 screened patients, 26 were included (17 with hypersensitivity, 9 switched to alternative asparaginase). Among hypersensitive patients, three had prior SAA levels: two showed levels below the therapeutic threshold (<0.100 IU/mL) before reactions, suggesting predictive potential. No significant associations were found between hypersensitivity and gender (p=0.27), ethnicity (p=0.49), race (p=0.22), or dose capping (p=1.0). Conclusions: Low SAA levels may indicate impending hypersensitivity or neutralizing antibody formation, supporting therapeutic drug monitoring. Patient-specific factors did not predict hypersensitivity. Larger, prospective studies are needed to validate these findings. Keywords: Calaspargase, Hypersensitivity, Serum Asparaginase Activity, Acute Lymphoblastic Leukemia, Therapeutic Drug Monitoring