Protoplasts are plant cells whose cell walls have been removed by enzymatic or mechanical means, and are used mainly for in vitro genetic engineering experiments. This chapter presents a detailed protocol for isolating protoplasts from the mesophyll cells of A. thaliana. We also describe applications of these protoplasts, including subcellular localization of protein, bimolecular fluorescence complementation assays for protein-protein interactions, and the application of exogenous hormones in protoplasts isolated from reporter lines such as TCSn::GFP and DR5::GFP. The general procedure includes protoplast isolation, transfection with the plasmids of interest, and visualization under confocal microscopy. This protocol is routinely used in our laboratory, primarily for protein-protein interaction studies.