Glutathione (GSH) is a sulfur-containing tripeptide composed of glutamic acid, cysteine, and glycine, and is a major non-protein thiol in many organisms, including plants. It plays a crucial role in redox homeostasis and signaling, as well as in defense against biotic and abiotic stresses. The functions of GSH are multifaceted, including buffering redox potential, scavenging reactive oxygen species (ROS), and integrating environmental information into cellular networks. The status of GSH is modulated by oxidants, nutritional factors, and other environmental cues, influencing protein structure and activity through changes in the thiol-disulfide balance. The historical overview of GSH in plants traces its discovery from yeast to plant tissues, highlighting its role in reducing oxidized forms of ascorbic acid and its importance in stress resistance. The biochemical and redox properties of GSH are discussed, including its structure, distribution, and redox reactions. Techniques for measuring GSH in plants, such as spectrophotometric assays and liquid chromatography-based methods, are described, along with their limitations and applications. The multiple functions of GSH in plants are explored, including its role in glutathione S-transferases, glyoxylase and formaldehyde metabolism, phytochelatins and heavy metal resistance, and sulfur assimilation. The regulation and manipulation of GSH synthesis are also covered, focusing on the enzymes involved, mutants, and inhibitors. The synthesis of GSH is regulated at multiple levels, with key controls including γ-glutamylcysteine synthetase (γ-ECS) activity and cysteine concentration. Overexpression of GSH synthesis genes and the use of butionine sulfoximine (BSO) as an inhibitor are discussed, along with the identification of chloroplast γ-ECS and GSH transporters. Overall, the chapter provides a comprehensive overview of the components that regulate GSH homeostasis and its roles in physiological processes such as light signaling, cell death, and defense against microbial pathogens and herbivores.Glutathione (GSH) is a sulfur-containing tripeptide composed of glutamic acid, cysteine, and glycine, and is a major non-protein thiol in many organisms, including plants. It plays a crucial role in redox homeostasis and signaling, as well as in defense against biotic and abiotic stresses. The functions of GSH are multifaceted, including buffering redox potential, scavenging reactive oxygen species (ROS), and integrating environmental information into cellular networks. The status of GSH is modulated by oxidants, nutritional factors, and other environmental cues, influencing protein structure and activity through changes in the thiol-disulfide balance. The historical overview of GSH in plants traces its discovery from yeast to plant tissues, highlighting its role in reducing oxidized forms of ascorbic acid and its importance in stress resistance. The biochemical and redox properties of GSH are discussed, including its structure, distribution, and redox reactions. Techniques for measuring GSH in plants, such as spectrophotometric assays and liquid chromatography-based methods, are described, along with their limitations and applications. The multiple functions of GSH in plants are explored, including its role in glutathione S-transferases, glyoxylase and formaldehyde metabolism, phytochelatins and heavy metal resistance, and sulfur assimilation. The regulation and manipulation of GSH synthesis are also covered, focusing on the enzymes involved, mutants, and inhibitors. The synthesis of GSH is regulated at multiple levels, with key controls including γ-glutamylcysteine synthetase (γ-ECS) activity and cysteine concentration. Overexpression of GSH synthesis genes and the use of butionine sulfoximine (BSO) as an inhibitor are discussed, along with the identification of chloroplast γ-ECS and GSH transporters. Overall, the chapter provides a comprehensive overview of the components that regulate GSH homeostasis and its roles in physiological processes such as light signaling, cell death, and defense against microbial pathogens and herbivores.