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Thousands of nanomaterials (NMs) are in commerce and few have toxicity data. To prioritize NMs for toxicity testing, high-throughput screening (HTS) of biological activity may be the only practical and timely approach to provide the necessary information. As in all nanotoxicologic studies, characterization of physicochemical properties of NMs and their immediate environments in HTS is critical to understanding how these properties affect NM bioactivity and to allow extrapolation to NMs not screened. The purpose of the study, the expert-groups-recommended minimal characterization, and NM physicochemical properties likely to affect measured bioactivity all help determine the scope of characterization. A major obstacle in reaping the full benefits of HTS for NMs is the low throughput of NM physicochemical characterization, which may require more sample quantity than HTS assays. Increasing the throughput and speed, and decreasing the amount of NMs needed for characterization are crucial. Finding characterization techniques and biological activity assays compatible with diverse classes of NMs is a challenge and multiple approaches for the same endpoints may be necessary. Use of computational tools and nanoinformatics for organizing and analyzing data are important to fully utilize the power of HTS. Other desired advances include the ability to more fully characterize: pristine NM without prior knowledge of NM physicochemical properties; non-pristine NMs (e.g., after use); NM in not-perfectly-dispersed suspension; and NM in biological samples at exposure-relevant conditions. Through combining HTS and physicochemical characterization results, we will better understand NM bioactivities, prioritize NMs for further testing, and build computational models to predict NM toxicity. WIREs Nanomed Nanobiotechnol 2013. doi: 10.1002/wnan.1229 Conflict of interest: The authors have declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website.