Background: The basophil activation test (BAT) using CD63 expression is a sensitive and specific tool for the diagnostic workup of autoimmune chronic spontaneous urticaria (CSU). The definition of a positive BAT is directly dependent on the reference range and the cutoff values established in control populations. As of now, the pediatric reference range and cutoff values of the CD63 BAT remain to be established. Methods: In this study, we analyzed CD63 expression in 80 children (1-17 years old) without chronic urticaria (i.e., controls) and compared the values to those of a pediatric cohort of 105 CSU patients and 23 physical urticaria (PU) patients. Results: Based on the log-normal distribution of CD63 values in control subjects, the reference range and the cutoff for positive CD63 BAT values was established to be 1.2-1.8% (95% CI) and 1.8%, respectively. Children with CSU showed significantly elevated and significantly increased BAT values compared to healthy controls (Wilcoxon rank test p value <0.001). In contrast, no difference was found between BAT results in controls and PU patients. In pediatric CSU patients, a higher disease activity was associated with higher BAT values. Conclusions: Our study provides, for the first time, reference and cutoff values for the CD63 BAT in children. Our findings show that positive CD63 BAT are common in children with CSU and linked to a high disease activity.