Nanotoxicology, a new branch of bionanoscience, deals with the study and application of the toxic or biological effects of nanomaterials or nanostructures, and aims to fill gaps in our knowledge of interactions between nano- and biosystems. However, progress in this new discipline largely relies on developing methodology to characterize nanomaterials in biological samples, quantify nanoparticles in living systems, and study their uptake, translocation, biodistribution, location and chemical status in vitro and in vivo, etc. In this review article, we focus on the main features of synchrotron radiation-based methods and their application to the study of the toxicological activities of nanomaterials. Synchrotron radiation-based analytical techniques are shown to provide a potent means for characterizing the toxic or biological behaviors of nanoparticles in biological systems.