Background: Dupilumab has proven to be an effective and safe treatment for atopic dermatitis (AD) in pediatric patients in clinical trials. However, few daily practice outcomes are available. Objectives: To evaluate the effect of 16 weeks dupilumab treatment on effectiveness, safety, and serum biomarkers in pediatric patients with moderate-to-severe AD in daily practice. Method: Patients visited the outpatient clinic at baseline, and after 4 and 16 weeks of treatment. Endpoints were proportions of patients achieving ≥50%, ≥75% or ≥90% improvement in Eczema Area and Severity Index (EASI), (almost) clear on Investigator Global Assessment (IGA) and ≥4 points reduction in Patient-Oriented Eczema Measure (POEM), Numeric Rating Scale (NRS)-pruritus, and -pain score at 16 weeks. Patients achieving absolute cutoff scores indicating controlled disease (EASI ≤7, POEM ≤7, NRS-pruritus ≤4) were analyzed. Nineteen severity-associated serum biomarkers were measured using Luminex-based multiplex immunoassays, and predicted-EASI (p-EASI) was calculated. Results: Forty-seven patients were included. Respectively 76.6%, 40.4%, and 19.1% reached EASI-50, EASI-75 and EASI-90, and 25.5% achieved an IGA-score (almost) clear. Improvement of ≥4 points on POEM, NRS-pruritus, and NRS-pain score was reached by 65.2%, 50.0%, and 79.2%, respectively. After 16 weeks, 83.0% achieved ≥1 cutoff score indicating controlled disease. Most frequently reported side effects were conjunctivitis (n=3 (6.4%)), hair loss (n=3) and headache (n=3). Biomarkers TARC, PARC, periostin, sIL-2Ra, and eotaxin-3 significantly decreased during treatment. The p-EASI showed a significantly high correlation with disease severity. Conclusion: Dupilumab treatment significantly improved disease severity and decreased severity-associated serum biomarkers in pediatric AD patients in daily practice.