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Introduction: Eosinophil‐derived neurotoxin (EDN) is a biomarker for eosinophilic activation. Urinary (u) EDN may allow non‐invasive monitoring of asthma, but clinical recommendations are lacking. We assessed the potential of uEDN as a marker of disease activity in pediatric asthma. Method s : We assessed urine samples of 371 children from the German ALLIANCE study cohort, from which we had: 169 preschool wheezers (<6 years), 80 asthmatics (≥6 years), and 122 healthy controls using the ImmunoCAP™ EDN Assay. Creatinine (Cr)‐adjusted uEDN values were analyzed using correlations, association tests, (non) parametric statistics, multiple linear, and multivariable regression. Results: uEDN/uCr values were higher in atopic versus non‐atopic preschool‐aged subjects (p =.035) and associated with the sum of allergen‐specific IgE in younger (r = 0.24, p =.003), and older subjects (r = 0.23, p =.043). uEDN/uCr was marginally a good determinant for atopy (p =.078, for subjects aged <6 years, and p =.058 for subjects ≥6 years). Children with the T2‐high phenotype had higher uEDN/uCr (p <.001) versus T2‐low—irrespective of using uEDN/uCr or blood eosinophils in combination to allergen sIgE for disease phenotyping. uEDN/uCr significantly correlated with reduced lung function among asthmatics (FEV1 z‐scores: r = −0.30, p =.007, and FEV1/FVC z‐scores: r = −0.24, p =.038). Using multivariable modeling, uEDN/uCr was an independent determinant of FEV1 (p =.038), and to a lesser extent, FEV1/FVC (p =.080). Conclusions: uEDN/uCr may serve as a non‐invasive biomarker for clinical features such as lung function in pediatric asthma. We highlight the utility of uEDN/uCr as a biomarker that can be easily assessed using widely available robust diagnostic immunoassays. [ABSTRACT FROM AUTHOR]