Peroxynitrite is the product of the nearly diffusion limited reaction between two free radicals, nitric oxide and superoxide. Peroxynitrite is a relatively long-lived, highly reactive species that can undergo harmful secondary oxidations of key cellular components. The reactivity of peroxynitrite is highly pH dependent and is influenced by the presence of metal, thiols, and bicarbonate. The products of some peroxynitrite reactions are indistinguishable from those derived form other reactive oxygen species and these products cannot be used for the detection of peroxynitrite in vivo. The detection of peroxynitrite in vivo is further comlicated by the multiple pathways of peroxynitrite reactivity with different biological molecules. Based on our current understanding of the chemical reactivity of peroxynitrite, methods for its detection are discussed.