Current diagnostic methods for JIA lack specificity, often failing to distinguish it from other childhood diseases of similar clinical presentations. The present study is a comparative cross-sectional study that identified potential biomarkers using label-free mass spectrometry and bioinformatics. Two differentially expressed proteins (DEPs), Myosin light chain 12b (Myl12b) and Mannose-binding lectin serine protease 1 (MASP1), showed increased expression in JIA patients. Receiver operating characteristic (ROC) analysis revealed strong predictive power (AUC: Myl12b = 0.757, MASP1 = 0.855), validated in a separate cohort via Western blot and ELISA. These findings suggest Myl12b and MASP1 as promising biomarkers for JIA diagnosis and treatment. Data: ProteomeXchange (PXD058863).