Nanotechnology is a key enabling technology. Still existing uncertainties concerning EHS need to be addressed to explore the full potential of this new technology. One challenge consists in the development of methods that reliably identify, characterize and quantify nanomaterials (NM) both as substance and in various products and matrices. The European Commission has recently recommended a definition of NM as reference to determine whether an unknown material can be considered as 'nanomaterial' (2011/696/EU). The proposed NanoDefine project will explicitly address this question. A consortium of European top RTD performers, metrology institutes and nanomaterials and instrument manufacturers has been established to mobilize the critical mass of expertise required to support the implementation of the definition. Based on a comprehensive evaluation of existing methodologies and a rigorous intra-lab and inter-lab comparison, validated measurement methods and instruments will be developed that are robust, readily implementable, cost-effective and capable to reliably measure the size of particles in the range of 1–100 nm, with different shapes, coatings and for the widest possible range of materials, in various complex media and products. Case studies will assess their applicability for various sectors, including food/feed, cosmetics etc. One major outcome of the project will be the establishment of an integrated tiered approach including validated rapid screening methods (tier 1) and validated in depth methods (tier 2), with a user manual to guide end-users, such as manufacturers, regulatory bodies and contract laboratories, to implement the developed methodology. NanoDefine will be strongly linked to main standardization bodies, such as CEN, ISO and OECD, by actively participating in TCs and WGs, and by proposing specific ISO/CEN work items, to integrate the developed and validated methodology into the current standardization work.