A new yellow fluorescent protein, Citrine, has been developed that is less sensitive to environmental factors such as pH and chloride ions compared to previous yellow fluorescent proteins (YFPs). Citrine was created through protein evolution in *E. coli* and features a mutation (Q69M) that lowers its pKa to 5.7, making it less sensitive to pH changes and more resistant to chloride interference. It also exhibits twice the photostability of previous YFPs and better expression at 37°C and in organelles. The structural explanation for these improvements involves the methionine side chain filling a previously large halide-binding cavity adjacent to the chromophore. Citrine has been used to create improved calcium indicators by fusing it with calmodulin or other proteins. These chimeras can be targeted to multiple cellular locations and have enabled the first single-cell imaging of free [Ca²⁺] in the Golgi. Citrine is superior to all previous YFPs except when pH or halide sensitivity is desired. It is particularly advantageous in genetically encoded fluorescent indicators of physiological signals. The study describes the development of Citrine, its structural and spectroscopic properties, and its application in creating improved calcium indicators. The x-ray structure of Citrine was determined, revealing how the Q69M mutation enhances its stability and reduces sensitivity to environmental factors. The results show that Citrine is more stable, less sensitive to pH and chloride, and better expressed in organelles compared to previous YFPs. These properties make Citrine a valuable tool for studying intracellular calcium dynamics and other physiological signals.A new yellow fluorescent protein, Citrine, has been developed that is less sensitive to environmental factors such as pH and chloride ions compared to previous yellow fluorescent proteins (YFPs). Citrine was created through protein evolution in *E. coli* and features a mutation (Q69M) that lowers its pKa to 5.7, making it less sensitive to pH changes and more resistant to chloride interference. It also exhibits twice the photostability of previous YFPs and better expression at 37°C and in organelles. The structural explanation for these improvements involves the methionine side chain filling a previously large halide-binding cavity adjacent to the chromophore. Citrine has been used to create improved calcium indicators by fusing it with calmodulin or other proteins. These chimeras can be targeted to multiple cellular locations and have enabled the first single-cell imaging of free [Ca²⁺] in the Golgi. Citrine is superior to all previous YFPs except when pH or halide sensitivity is desired. It is particularly advantageous in genetically encoded fluorescent indicators of physiological signals. The study describes the development of Citrine, its structural and spectroscopic properties, and its application in creating improved calcium indicators. The x-ray structure of Citrine was determined, revealing how the Q69M mutation enhances its stability and reduces sensitivity to environmental factors. The results show that Citrine is more stable, less sensitive to pH and chloride, and better expressed in organelles compared to previous YFPs. These properties make Citrine a valuable tool for studying intracellular calcium dynamics and other physiological signals.