Rationale Biomarkers predicting development of atopic disease are needed for targeted preventive measures and to study if disease pathology may be active prior to onset of symptoms. Objective To investigate whether eosinophil protein-X, leukotriene-C4/D4/E4 and 11ß-prostaglandin-F2a (prostaglandin D2 metabolite) assessed in urine from healthy at-risk neonates precede development of atopic disease during the first 6 years of life. Methods We measured eosinophil protein-X (N=369), leukotriene-C4/D4/E4 (N=367), and 11ß-prostaglandin-F2a (N=366) in urine from 1-month-old children participating in the Copenhagen Prospective Studies on Asthma in Childhood (COPSAC) birth cohort. Clinical data on development of allergic sensitization, allergic rhinitis, nasal eosinophilia, blood eosinophilia, eczema, troublesome lung symptoms (significant cough or wheeze or dyspnoea) and asthma were collected prospectively untill age 6 years. Associations between urinary biomarkers and development of atopic traits were investigated using general estimating equations, logistic regression and Cox regression. Measurements and Main Results Eosinophil protein-X in the urine of the asymptomatic 1-month-old neonates was significantly associated with development of allergic sensitization (Odds ratio, 1.49; 95% CI, 1.08-1.89), nasal eosinophilia (Odds ratio, 3.2; 95% CI, 1.2-8.8), and eczema (Hazard ratio, 1.4; 95% CI, 1.0-2.0), but not with allergic rhinitis, asthma or blood eosinophilia. Neither leukotriene-C4/D4/E4 nor 11ß-prostaglandin-F2a was associated with any of the atopic phenotypes. Conclusion Eosinophil protein-X in urine from asymptomatic neonates is a biomarker significantly associated with later development of allergic sensitization, nasal eosinophilia and eczema during the first 6 years of life. These findings suggest activation of eosinophil granulocytes early in life prior to development of atopy-related symptoms.