Diabetic retinopathy (DR) is the major cause of acquired blindness in working-age adults. Current treatments for DR (laser photocoagulation, intravitreal corticosteroids, intravitreal antivascular endothelial growth factor (VEGF) agents, and vitreo-retinal surgery) are applicable only at advanced stages of the disease and are associated with significant adverse effects. Therefore, new pharmacological treatments for the early stages of the disease are needed. Vitreous fluid obtained from diabetic patients undergoing vitreoretinal surgery is currently used to explore the events that are taking place in the retina for clinical research. However, several confounding factors such as vitreous haemorrhage and concentration of vitreous proteins should be considered in the analysis of the results. In this paper we will focus on the vitreous fluid as a tool for exploring the mediators of DR and in particular the molecules related to inflammatory pathways. In addition, their role in the pathogenesis of DR will be discussed. The usefulness of new technologies such as flow cytometry and proteomics in identifying new candidates involved in the inflammatory process that occurs in DR will be overviewed. Finally, a more personalized treatment based on vitreous fluid analysis aiming to reduce the burden associated with DR is suggested.