Background: Asparaginase (ASNase) is an important anti‐leukemic drug in the treatment of childhood acute lymphoblastic leukemia (ALL) and non‐Hodgkin lymphoma (NHL). Depletion of asparagine by ASNase results in selective apoptosis of lymphoblasts which depend on an external source of asparagine for their cell growth. A substantial proportion of patients develops anti‐ASNase neutralising antibodies, resulting in allergic reactions or silent inactivation (SI), characterized by rapid clearance and inactivation of ASNase. Aims: Prospective, real‐time therapeutic drug monitoring of peg‐ASNase (Oncaspar ® ) and Erwinia ASNase (Erwinase ® ) in children treated for ALL and NHL in Belgium. Methods: Patients (1‐18y) with newly diagnosed ALL and precursor B‐ or T‐lymphoblastic NHL from 8 Belgian pediatric hemato‐oncology centres were enrolled between 01/2013 and 11/2017. All patients were treated according to the treatment guidelines of the EORTC‐CLG 58081 study. ASNase activity was quantified using the AHA test (described by Lanvers et al., 2002) using a SpectraMax M3 spectrophotometer (Molecular Devices). ASNase activity >100U/L was considered to be sufficient for complete depletion of asparagine. Erwinia ASNase was given as second line after allergic reaction or silent inactivation to Peg‐ASNase. One dose of Peg‐ASNase 2,500IU/m 2 was replaced by 6 doses of 20,000U/m 2 Erwinia ASNase in 2 weeks. Results: In total, 286 children (118 girls and 168 boys) with newly diagnosed ALL (n = 260) and NHL (n = 26) were enrolled in the prospective real‐time ASNase monitoring programme. Clinical allergic reactions were seen in 33 (11.5%), and silent inactivation in 14 (4.9%) patients treated with peg‐ASNase. Most allergies were CTCAE 4.03 grade 2‐3 and occurred after the second or third administration. SI was mainly seen after the second administration and in maintenance therapy. Patients were more at risk for hypersensitivity reactions after an ASNase‐free period. Forty‐two of them were switched to Erwinia ASNase. Three patients ...