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). It discusses the use of probes such as dichlorodihydrofluorescein diacetate (DCFH-DA), hydroethidine (HE), and dihydrorhodamine (DHR) for measuring hydrogen peroxide (H₂O₂), superoxide (O₂⁻), and peroxynitrite (ONOO⁻). While these probes are widely used, they have significant limitations. For example, DCFH-DA is not a direct measure of H₂O₂ but can be oxidized by various one-electron oxidants, leading to false positive signals. Similarly, HE and Mito-SOX produce red fluorescence that is not a reliable indicator of superoxide due to the formation of 2-hydroxyethidium (2-OH-E⁺) and 2-hydroxymitoethidium (2-OH-Mito-E⁺), which have overlapping fluorescence spectra. DHR is also not specific for ONOO⁻, as it can be oxidized by various oxidants, including ·NO and O₂⁻. The paper recommends alternative probes and analytical techniques to improve the accuracy of ROS and RNS measurements. It also highlights the importance of using appropriate inhibitors and other methods to distinguish between different oxidants. Emerging probes, such as aromatic boronates, offer more specific detection of H₂O₂ and ONOO⁻. The paper emphasizes the need for careful interpretation of results and the use of multiple techniques to ensure accurate measurement of ROS and RNS in biological systems.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). It discusses the use of probes such as dichlorodihydrofluorescein diacetate (DCFH-DA), hydroethidine (HE), and dihydrorhodamine (DHR) for measuring hydrogen peroxide (H₂O₂), superoxide (O₂⁻), and peroxynitrite (ONOO⁻). While these probes are widely used, they have significant limitations. For example, DCFH-DA is not a direct measure of H₂O₂ but can be oxidized by various one-electron oxidants, leading to false positive signals. Similarly, HE and Mito-SOX produce red fluorescence that is not a reliable indicator of superoxide due to the formation of 2-hydroxyethidium (2-OH-E⁺) and 2-hydroxymitoethidium (2-OH-Mito-E⁺), which have overlapping fluorescence spectra. DHR is also not specific for ONOO⁻, as it can be oxidized by various oxidants, including ·NO and O₂⁻. The paper recommends alternative probes and analytical techniques to improve the accuracy of ROS and RNS measurements. It also highlights the importance of using appropriate inhibitors and other methods to distinguish between different oxidants. Emerging probes, such as aromatic boronates, offer more specific detection of H₂O₂ and ONOO⁻. The paper emphasizes the need for careful interpretation of results and the use of multiple techniques to ensure accurate measurement of ROS and RNS in biological systems.