Updated project metadata. Congenital diaphragmatic hernia (CDH) results from incomplete diaphragm development and can be associated with pulmonary hypoplasia, pulmonary hypertension, and heart failure. Currently, few biomarkers are available. Since amniotic fluid comprises proteins of both fetal and maternal origin, its analysis could provide insights on mechanisms underlying CDH and provide biomarkers for early diagnosis, severity of pulmonary changes and response to treatment. The study objective was to identify proteomic changes in amniotic fluid consistently associated with CDH. Amniotic fluid was obtained at term (37-39 weeks) from women with normal pregnancies (n=5) or carrying fetuses with CDH (n=5). After immuno-depletion of the highest abundance proteins, off-line fractionation and high-resolution tandem mass spectrometry were performed and quantitative differences between the proteomes of the groups were determined. Of 1036 proteins identified, 218 were differentially abundant. Bioinformatics analysis showed significant changes in GP6 signaling, MSP-RON signaling in macrophages pathways and networks associated with cardiovascular system development and function, connective tissue disorders and dermatological conditions. Pulmonary surfactant protein B, osteopontin, kallikrein 5 and galectin 3 were investigated orthogonally using ELISA in larger cohorts to test the observed differences and showed statistically significant differences aiding in the prediction of CDH. The findings provide potential tools for improved clinical detection and management of CDH.