<b><i>Introduction:</i></b> Current techniques to diagnose and/or monitor critically ill neonates with bronchopulmonary dysplasia (BPD) require invasive sampling of body fluids, which is suboptimal in these frail neonates. We tested our hypothesis that it is feasible to use noninvasively collected urine samples for proteomics from extremely low gestational age newborns (ELGANs) at risk for BPD to confirm previously identified proteins and biomarkers associated with BPD. <b><i>Methods:</i></b> We developed a robust high-throughput urine proteomics methodology that requires only 50 μL of urine. We utilized the methodology with a proof-of-concept study validating proteins previously identified in invasively collected sample types such as blood and/or tracheal aspirates on urine collected within 72 h of birth from ELGANs (gestational age [26 ± 1.2] weeks) who were admitted to a single Neonatal Intensive Care Unit (NICU), half of whom eventually developed BPD (<i>n</i> = 21), while the other half served as controls (<i>n</i> = 21). <b><i>Results:</i></b> Our high-throughput urine proteomics approach clearly identified several BPD-associated changes in the urine proteome recapitulating expected blood proteome changes, and several urinary proteins predicted BPD risk. Interestingly, 16 of the identified urinary proteins are known targets of drugs approved by the Food and Drug Administration. <b><i>Conclusion:</i></b> In addition to validating numerous proteins, previously found in invasively collected blood, tracheal aspirate, and bronchoalveolar lavage, that have been implicated in BPD pathophysiology, urine proteomics also suggested novel potential therapeutic targets. Ease of access to urine could allow for sequential proteomic evaluations for longitudinal monitoring of disease progression and impact of therapeutic intervention in future studies.