Recombinant adeno-associated virus (AAV) vectors have been widely used in gene therapy applications for central nervous system (CNS) diseases and have yielded favorable preclinical and clinical outcomes. These vectors can successfully transduce post-mitotic cells, such as neurons and astrocytes. Emerging evidence indicates that the microglia, the major resident immune cells in the CNS, play a central role in the pathobiology of neurodegenerative disorders. Consequently, these cells have recently become the focus of new treatment approaches. However, these approaches have been hindered by the difficulty in transducing microglia using viral and non-viral vectors. To address this, here we introduce a novel microglia culture protocol from mouse retina, which is reproducible and is characterized by high cell numbers and long in vitro viability. We used this model as a screening platform to evaluate CMV-mediated reporter transgene expression of engineered AAV6- or AAV1-derived capsids, the most used serotypes on glial cells. The variants carrying 12-mer peptide insertions, AAV-GL and AAV-GL.R, showed enhanced transduction efficiency of primary mouse retinal microglia cells compared with not only wild-type but also the recently published triple mutant AAV6 TM. This improved activity has been confirmed in both resting and chemically activated microglia, mimicking in vivo degenerating settings. Finally, we started to validate our variants in mouse models of retinal disorders. Overall, our study establishes a new in vitro platform for assessing microglial transduction and identifying novel AAV capsids with improved microglial transduction properties.