Nanotechnology offers many advantages to traditional drug design, delivery and medical diagnostics; however, nanomedicines present considerable challenges for preclinical development. Nanoparticle constructs intended for medical applications consist of a wide variety of materials, and their small size, unique physicochemical properties and biological activity often require modification of standard characterization techniques. A rational characterization strategy for nanomedicines includes physicochemical characterization, sterility and pyrogenicity assessment, biodistribution (absorption, distribution, metabolism and excretion [ADME]) and toxicity characterization, which includes both in vitro tests and in vivo animal studies. Here, we highlight progress for a few methods that are uniquely useful for nanoparticles or are indicative of their toxicity or efficacy.