This study was conducted to identify the contribution of myositis-specific antibodies (MSAs) on the correlation between type I interferon (IFN-I) plasma levels and disease activity in juvenile dermatomyositis (JDM) patients. We prospectively obtained 198 samples from 49 JDM patients. Autoantibody levels were determined for all patients. Muscle activity was scored using Childhood Myositis Assessment Scale (CMAS) and Manual Muscle Testing in 8 muscles (MMT-8) and skin involvement with a Disease Activity Score (DAS). Three homebrew digital ELISA measuring respectively the IFN-α2 subtype, all 12 interferon alpha (IFN-α) subtype proteins (Pan-α), and interferon beta (IFN-β) were used to quantify IFN-I in patient’s plasma. Correlations between IFN-I titers and clinical scores were evaluated using linear mixed-effect models. In our entire cohort, Pan-α and IFN-β were associated with all three clinical scores. Correlations between IFN-β plasma levels and muscle disease activity were stronger than those with IFN-α levels. After stratification on MSAs expression, we found a relatively equal distribution for the most frequent MSAs phenotypes (MDA5+:n=13, NXP2+:n=12, TIF1g+:n=11, No MSAs:n=13). We observed an association between CMAS and IFN-β in all 4 subgroups. In contrast, Pan-α levels were associated with clinical scores only in MDA5+ patients. In this subgroup, IFN-α2 was also strongly associated with muscle scores. These findings underscore the potential role of IFN-β in blood as a reliable biomarker of muscle disease activity in JDM, irrespectively of MSAs positivity. The association of IFN-α subtypes plasma levels with clinical scores only in MDA5+ patients suggests a distinct pathophysiological mechanism in this subgroup.