Real‐time quantification of reactive nitrogen and oxygen species (ROS) in cells is of paramount importance as they are essential for cellular functions. Their excessive formation contributes to the dysfunction of cells and organisms, ultimately leading to cell death. As ROS are mostly produced in the mitochondria, we have synthesized a fluorescent probe able to reach this organelle to detect and quantify, in real time, the variation of ROS by time‐resolved microfluorimetry. The new probes are based on the long fluorescence lifetime of pyrene butyric acid (PBA). Two PBA isomers, attached at their 1‐ or 2‐positions to a peptide vector to target mitochondria, were compared and were shown to allow the measurement of free radical species and oxygen, but not non‐radical species such as H2O2.

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