Single-cell RNA sequencing (scRNA-seq) is an emerging technology that can address the challenge of cellular heterogeneity. In the last decade, the cost per cell has been dramatically reduced, and the throughput has been increased by 10-fold. Like many other tissues, the retina is highly heterogeneous with an estimated of over 100 subtypes of neuronal cells. Here, we describe the current techniques to perform scRNA-seq on the adult retinal tissue including retinal dissection, retinal dissociation, assessment of cell population, cDNA synthesis, library construction, and next-generation sequencing. In addition, we introduce a workflow of scRNA-seq data analysis using open-source tools.