Proliferative diabetic retinopathy (PDR) is the most severe form of diabetic retinopathy and, along with diabetic macular edema, is responsible for the majority of blindness in adults below the age of 65. Therapeutic strategies for PDR are ineffective at curtailing disease progression in all cases; however a deeper understanding of the ocular metabolic landscape in PDR through metabolomic analysis may offer new therapeutic targets. Here, global and targeted mass spectrometry-based metabolomics were used to investigate metabolism. Initial analyses on vitreous humor from patients with PDR (n = 9) and non-diabetic controls (n = 11) revealed an increase of arginine and acylcarnitine metabolism in PDR. The oxygen-induced-retinopathy (OIR) mouse model, which exhibits comparable pathological manifestations to human PDR, revealed similar increases of arginine and other metabolites in the urea cycle, as well as downregulation of purine metabolism. We validated our findings by targeted multiple reaction monitoring and through the analysis of a second set of patient samples [PDR (n = 11) and non-diabetic controls (n = 20)]. These results confirmed a predominant and consistent increase in proline in both the OIR mouse model and vitreous samples from patients with PDR, suggesting that over activity in the arginine-to-proline pathway could be used as a therapeutic target in diabetic retinopathy.