This position paper critically analyzes the challenges and limitations of commonly used fluorescent probes for detecting and measuring reactive oxygen and nitrogen species (ROS and RNS). The authors discuss the specific products formed from the reactions between these probes and ROS/RNS, and provide recommendations for alternative probes and analytical techniques. They emphasize the importance of accurate detection and characterization of ROS/RNS to attribute specific cell signaling events to particular species. The paper highlights the complex intracellular redox chemistry of probes like dichlorodihydrofluorescein diacetate (DCFH-DA) and hydroethidine, and their limitations in measuring intracellular H₂O₂ and superoxide. It also introduces emerging probes, such as boronate-containing fluorophores, which can more specifically detect peroxynitrite. The authors recommend guidelines for the optimal use of selected fluorescent probes and the interpretation of results, aiming to clarify the appropriate use of these probes in biological research.This position paper critically analyzes the challenges and limitations of commonly used fluorescent probes for detecting and measuring reactive oxygen and nitrogen species (ROS and RNS). The authors discuss the specific products formed from the reactions between these probes and ROS/RNS, and provide recommendations for alternative probes and analytical techniques. They emphasize the importance of accurate detection and characterization of ROS/RNS to attribute specific cell signaling events to particular species. The paper highlights the complex intracellular redox chemistry of probes like dichlorodihydrofluorescein diacetate (DCFH-DA) and hydroethidine, and their limitations in measuring intracellular H₂O₂ and superoxide. It also introduces emerging probes, such as boronate-containing fluorophores, which can more specifically detect peroxynitrite. The authors recommend guidelines for the optimal use of selected fluorescent probes and the interpretation of results, aiming to clarify the appropriate use of these probes in biological research.