Proliferative diabetic retinopathy (PDR) remains a leading cause of blindness despite progress in screening and treatment. Recently, the Warburg effect, a metabolic alteration affecting amino acid (AA) metabolism in proliferating cells, has drawn attention regarding its role in PDR. This study aimed to investigate the impact of the Warburg effect on AA metabolism in human retinal endothelial cells (HRECs) subjected to PDR-associated risk factors and validate the findings in patients with PDR. In vitro experiments exposed HRECs to high glucose (HG) and/or hypoxia (Hyp), known inducers of the Warburg effect. The HG + Hyp group of HRECs exhibited significant differences in non-essential AAs with aliphatic non-polar side chains, mainly driven by elevated glycine concentrations. Pathway enrichment analysis revealed several glycine metabolism-related pathways significantly altered due to the Warburg effect induced by HG + Hyp. Crucially, vitreous humor samples from PDR patients displayed higher glycine levels compared to non-diabetic and diabetic patients without PDR. The odds ratio for PDR patients with glycine levels above the cut-off of 0.0836 µM was 28 (p = 0.03) compared to non-PDR controls. In conclusion, this study provides mechanistic insights into how a specific Warburg effect subtype contributes to glycine accumulation in PDR and supports glycine's potential as a biomarker for PDR pathogenesis.