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Summary: Asparaginase (ASNase) is an important anti‐leukaemic drug in the treatment of childhood acute lymphoblastic leukaemia (ALL) and non‐Hodgkin lymphoma (NHL). A substantial proportion of patients develop hypersensitivity reactions with anti‐ASNase neutralising antibodies, resulting in allergic reactions or silent inactivation (SI), and characterised by inactivation and rapid clearance of ASNase. We report results of a prospective, real‐time therapeutic drug monitoring of pegylated Escherichia coli (PEG‐)ASNase and Erwinia ASNase in children treated for ALL and NHL in Belgium. Erwinia ASNase was given as second‐line after hypersensitivity to PEG‐ASNase. In total, 286 children were enrolled in the PEG‐ASNase cohort. Allergy was seen in 11·2% and SI in 5·2% of patients. Of the 42 patients treated with Erwinia ASNase, 7·1% experienced allergy and 2·4% SI. The median trough PEG‐ASNase activity was high in all patients without hypersensitivity. After Erwinia administration significantly more day 3 samples had activities <100 IU/l (62·5% vs. 10% at day 2 (D2)). The median D2 activity was significantly higher for intramuscular (IM; 347 IU/l) than for intravenous Erwinia administrations (159 IU/l). This prospective, multicentre study shows that monitoring of ASNase activity during treatment of children with ALL and NHL is feasible and informative. Treatment with Erwinia ASNase warrants close monitoring and optimally adherence to a 2‐day interval of IM administrations. [ABSTRACT FROM AUTHOR]