Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as the red fluorescent protein TagRFP. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H₂O₂) per molecule of chromophore. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H₂O₂, this study investigates the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Immature eGFP and TagRFP catalytically generate the free radical superoxide anion (O₂ •-) and H₂O₂ in the presence of NADH. Generation of the free radical O₂ •- and H₂O₂ by eGFP in the presence of NADH affects the gene expression of cells. Many biological pathways are altered, such as a decrease in HIF1α stabilization and activity. The biological pathways altered by eGFP are known to be implicated in the pathophysiology of many diseases associated with oxidative stress; therefore, it is critical that such experiments using fluorescent proteins are validated with alternative methodologies and the results are carefully interpreted. Since cells inevitably experience oxidative stress when fluorescent proteins are expressed, the use of this tool for cell labeling and in vivo cell tracing also requires validation using alternative methodologies.