Peroxynitrite, a representative of reactive nitrogen species, plays important roles in the physiological and pathological processes of many oxidative stress-related diseases. It is generated from the reaction of nitric oxide (NO) and superoxide (O2·-) and is far more active than its precursors. Peroxynitrite can be further decomposed into other cytotoxic reactive species. Peroxynitrite and its derivatives can interact with various biomolecules such as DNA and proteins. Due to its high reactivity and short lifetime, accurate detection of peroxynitrite in biological systems is a challenge task. In the last decade, huge efforts have been made to develop reliable techniques to assess the generation of peroxynitrite in various cellular and animal experiments. There are three major approaches for peroxynitrite detection, including electrochemical sensors, detection of nitrotyrosine formation, and fluorescent probes. Particularly, progress has been made in developing novel fluorescent probes to detect peroxynitrite with relatively high sensitivity and specificity. Herein, we review the recent progress made in peroxynitrite detection methods and discuss the advantages and disadvantages of these methods. The development of these techniques will offer new opportunities for understanding the roles of peroxynitrite in the oxidative stress-related physiological and pathological conditions and provide platforms for drug discovery targeting peroxynitrite and other free radicals for therapeutic purposes.