The successful translation of nanoparticle-based systems into commercial products depends upon the ability to reliably measure important physical and chemical properties of these particles. The density of nanoparticles is one such property, because it provides important information about the composition of the material. In this work, an analytical centrifugation approach based on line-start centrifugal sedimentation and flotation measurements is described. The two independent measurements permit both the size and the density of these nanoparticles to be determined with excellent precision. A set of monodisperse polystyrene nanoparticles of different sizes is used to demonstrate this method. The density and size measurements are validated by comparison to accurate Small Angle X-ray Scattering (SAXS) analysis for particles within the size range of SAXS, i.e. less than ∼300 nm in diameter. Both sedimentation and flotation measurements produce consistent high resolution size distributions of the particles and the measured size and density values are identical, within experimental uncertainty, to the SAXS results. This approach has the potential to provide useful characterisation of a range of particles of interest, for example, for medical application, such as liposomes and polymeric drug carriers.