In this study, we visualize and quantify retinal gliosis in vivo for monitoring early diabetic retinopathy (DR) in a transgenic mouse model. Onset of diabetes was triggered via intraperitoneal injection of streptozotocin (STZ) into transgenic F1 hybrid (FVB/N × C57BL/6J) mice expressing green fluorescent protein (GFP) under the control of glial fibrillary acidic protein (GFAP) promoter. Retinal glial cells are imaged once pre-STZ treatment followed by weekly post-STZ imaging for five weeks using a confocal scanning laser ophthalmoscope. Mice develop diabetes one week after STZ induction as confirmed from the high blood glucose levels (>13.9 mmol/L). A significant increase is observed in the GFAP-GFP transgene expression from astrocytic cell bodies and processes as early as week 5 for the STZ-treated mice. Retinal astrocytes also undergo hyperplasia progressively from week 0 to 5. This precedes any structural abnormalities to the retinal vasculature. Immunohistochemistry (IHC) on retinal sections as well as quantitative RT-PCR of endogenous and transgene GFAP mRNA supports our in vivo observation. Our in vivo data correlates with clinical reports with regards to retinal gliosis-related inflammatory response during early diabetic retinopathy. This opens up the possibility of using in vivo molecular imaging of retinal glial cells as a platform for monitoring the efficacy of anti-DR drug candidates which intervene at an early stage.