The protoplast transient assay system has been widely used for rapid functional analyses of genes using cellular and biochemical approaches. This system has been increasingly employed for functional genetic studies using double-stranded (ds) RNA interference (RNAi). Here, we describe a modified procedure for the isolation of protoplasts from leaf mesophyll cells of 14-day-old Arabidopsis thaliana. This modification significantly simplifies and speeds up functional studies without compromising the yield and the viability of protoplasts. We also present the procedure for the isolation and transfection of protoplasts from mesophyll cells of an emerging model grass species, Brachypodium distachyon. Further, we detail procedures for RNAi-based functional studies of genes using transient expression of in vitro synthesized dsRNA in protoplasts.