Arabidopsis mesophyll protoplasts provide a versatile system for transient gene expression analysis. This protocol describes a method for isolating protoplasts from Arabidopsis leaves, transfecting them with plasmid DNA using PEG-calcium, and culturing them to study gene function. The process takes 6–8 hours for isolation and DNA transfection, with results obtained in 2–24 hours. The system allows for cost-effective and efficient exploration of physiological responses and hypothesis-driven tests. Protoplasts retain many physiological responses of intact plants, making them suitable for studying signaling pathways, gene regulation, and cellular processes. The system is particularly useful for functional genomics studies in Arabidopsis, which has extensive genetic resources. Reporter genes such as luciferase and β-glucuronidase are used to quantify gene expression. The system also supports inducible gene expression using chemical inducers like dexamethasone. The protocol is applicable to various plant species and can be used to study both abiotic and biotic stress responses. It is also useful for analyzing protein interactions, localization, and trafficking. The system is efficient, requiring minimal laboratory equipment and resources, and allows for high-throughput analysis. However, it is not suitable for studying cell cycle regulation as protoplasts do not divide in the simple incubation buffer used. The system is also applicable to mutant plants to study gene function in different genetic backgrounds. The protocol includes detailed reagent preparation, equipment, and step-by-step procedures for protoplast isolation, transfection, and analysis. The system is widely used for studying plant hormone signaling, protein interactions, and gene regulation. The protocol has been tested and optimized for various Arabidopsis accessions and mutants. The system is a reliable and efficient tool for functional genomics research in plants.Arabidopsis mesophyll protoplasts provide a versatile system for transient gene expression analysis. This protocol describes a method for isolating protoplasts from Arabidopsis leaves, transfecting them with plasmid DNA using PEG-calcium, and culturing them to study gene function. The process takes 6–8 hours for isolation and DNA transfection, with results obtained in 2–24 hours. The system allows for cost-effective and efficient exploration of physiological responses and hypothesis-driven tests. Protoplasts retain many physiological responses of intact plants, making them suitable for studying signaling pathways, gene regulation, and cellular processes. The system is particularly useful for functional genomics studies in Arabidopsis, which has extensive genetic resources. Reporter genes such as luciferase and β-glucuronidase are used to quantify gene expression. The system also supports inducible gene expression using chemical inducers like dexamethasone. The protocol is applicable to various plant species and can be used to study both abiotic and biotic stress responses. It is also useful for analyzing protein interactions, localization, and trafficking. The system is efficient, requiring minimal laboratory equipment and resources, and allows for high-throughput analysis. However, it is not suitable for studying cell cycle regulation as protoplasts do not divide in the simple incubation buffer used. The system is also applicable to mutant plants to study gene function in different genetic backgrounds. The protocol includes detailed reagent preparation, equipment, and step-by-step procedures for protoplast isolation, transfection, and analysis. The system is widely used for studying plant hormone signaling, protein interactions, and gene regulation. The protocol has been tested and optimized for various Arabidopsis accessions and mutants. The system is a reliable and efficient tool for functional genomics research in plants.