In vivo redox reaction is involved in the processes of oxidative diseases. Thus, direct and non-invasive measurement of in vivo free radical reactions in living animals should be essential to understanding the roles of free radicals in pathophysiological phenomena. Electron spin resonance (ESR) technique has been utilized in analysis of free radicals, which are generated through imbalance of in vivo redox status. In vivo ESR/spin probe technique using nitroxide radicals as spin probes was developed for estimation of in vivo free radical reactions in whole living animals. This technique using a probe may become a powerful tool for use in clarifying mechanisms of disease and in monitoring pharmaceutical therapy. The application of ESR was introduced and discussed in this article.