Diabetic retinopathy (DR) is a leading cause of blindness in adults under 40 in the developed world, with a significant proportion progressing to vision-threatening stages like proliferative diabetic retinopathy (PDR) and neovascular glaucoma (NVG). This study aims to explore the molecular mechanisms underlying the progression from non-proliferative DR (NPDR) to PDR and NVG, focusing on identifying potential biomarkers and therapeutic targets. We utilized discovery-based proteomics, specifically label free quantification (LFQ), to analyze aqueous humor (AH) proteins from patients with NPDR, PDR, and NVG from the AH samples obtained during cataract surgery or anterior chamber paracentesis. We employed triple quadrupole (QQQ)-MS for targeted protein quantification to validate marker proteins for each disease state. Notably,subsequent analyses revealed specific proteins associated with each stage of DR. Gene ontology analysis and functional annotation unveiled significant processes in DR such as immune response and lipid metabolic process. Validation of potential biomarkers revealed proteins showing significant concentration changes, with several candidates demonstrating diagnostic potential through ROC curve analysis. These findings provide insights into the biomarkers and pathogenesis of these severe neovascular complications. The identification of specific proteins associated with the progression of DR to more advanced stages offer a promising avenue for early diagnosis and targeted therapy. The study underscores the potential of AH proteomics in understanding complex diseases like DR and its complications.