Advances in genomic and proteomic platforms enable high-throughput studies for regulatory factors and interactors involved in signaling network at a molecular level. However, it has never been trivial to verify the omics data in vivo or functionally integrate the data in a cell signaling context. For plants, genetic approaches using knockout mutants and transgenic lines have been mainly used to characterize functions of gene products in vivo. In general, such approaches demand a longer time and a higher cost and have difficulties in understanding gene functions comprehensively in a high-throughput manner. Transient gene expression is a method of choice to examine the cellular functions of genetic components in vivo. The leaf mesophyll protoplasts (LMP) provide a perfect system to transiently express a gene encoding an epitope-tagged protein of interest and quickly and reliably trace subcellular locales of the protein in a near high-throughput manner. Here, a simple and straightforward method for isolating leaf meshophyll protoplasts from Arabidopsis has been described in detail to help beginners initiate their first cell-based functional genomic analysis.