The specific and sensitive detection of peroxynitrite (ONOO–/ONOOH) in biological systems is a great challenge due to its high reactivity towards several biomolecules. Herein, we validated the advantages of using fluorescein-boronate (Fl-B) as a highly sensitive fluorescent probe for the direct detection of peroxynitrite under biologically-relevant conditions in two different cell models. The synthesis of Fl-B was achieved by a two-step conversion synthetic route with high purity (> 99 %) and overall yield (∼ 42%). Reactivity analysis of Fl-B with relevant biological oxidants were performed. The rate constant for the reaction of peroxynitrite with Fl-Bwas 1.7 x 106M-1s-1, a million times faster than the rate constant measured for H2O2 (k = 1.7 M-1s-1) and 2,700 faster than HOCl (6.2 x 102 M-1s-1) at 37° C and pH 7.4. The reaction of Fl-B with peroxynitrite was significant even in the presence of physiological concentrations of CO2, a well-known peroxynitrite reactant. Experimental and simulated kinetic analyses confirm that the main oxidation process of Fl-B takes place with peroxynitrite itself via a direct bimolecular reaction and not with peroxynitrite-derived radicals. Fl-B was successfully applied for the detection of endogenously- generated peroxynitrite by endothelial cells and in macrophage-phagocyted parasites. The generated data allowed estimating the actual intracellular flux of peroxynitrite, e.g., ionomycin-stimulated endothelial cells generated peroxynitrite at a rate of ∼ 0.1 μM s-1, while immunostimulated macrophages do so in the order of ∼ 1 μM s-1 inside T. cruzi-infected phagosomes. Cellular peroxynitrite detection was achieved by conventional laboratory fluorescence-based methods including flow cytometry and epi-fluorescence microscopy. Fl-B was shown to be more sensitive than the coumarin boronate due to a higher molar absorption coefficient and quantum yield. Overall, our results show that Fl-B is a kinetically selective and highly sensitive probe for the direct detection of cell-derived peroxynitrite. Moreover, the data allowed to tailor boronate-derivatives targeted to identify subcellular sites and rates of peroxynitrite generation; indeed, ongoing work is focused on the generation and characterization of new organelle-targeted boronate-based probes.