Peroxiredoxins catalytically reduce peroxynitrite to nitrite. The peroxidatic cysteine of peroxiredoxins reacts rapidly with peroxynitrite. The rate constant of that reaction can be measured using a stopped flow spectrophotometer either directly by following peroxynitrite disappearance in the region of 300 to 310 nm using an initial rate approach or steady-state measurements or by competition with a reaction of known rate constant. The reactions used to compete with peroxiredoxins include the oxidation of Mn(III)porphyrins and horseradish peroxidase by peroxynitrite. Additionally, a method is described in which a hydroperoxide competes with peroxynitrite for the oxidation of peroxiredoxin. Moreover, a fluorescent technique for determining the kinetics of thioredoxin-mediated peroxiredoxin reduction, closing the catalytic cycle, is also described. All methods reviewed provide reliable values of rate constants and a combination of them can be used to provide further reassurance; applicability and advantages of the different methodologies are discussed.