|Magnetic nanoparticles, especially those composed of iron oxide, are extremely attractive options when it comes to MRI contrast agents and magnetically targeted therapies. Their superparamagnetic properties as well as their high magnetic saturation and susceptibility make iron oxide MNPs excellent contrast agents for magnetic resonance imaging (MRI). In addition, their high surface-to-volume ratio enables a high loading of functionalities including imaging agents, targeting moieties, biocompatible ligands, and anticancer drugs. However, the inability to synthesize them in a monodisperse manner above ~20 nm in size has hindered their optimization for in vivo use. A novel and efficient approach to the size-selective fractionation of an original magnetic nanoparticle suspension into a number of more distinct size distributions has been developed and used to study the effect of size on the relaxometric properties of the particles. A series of experiments were conducted using particle suspensions of hydrodynamic diameters of 96.3 ± 9.0, 123.6 ± 7.9, and 141.5 ± 10.8 nm obtained from an original polydisperse suspension of nanoparticles to determine the effect of size on the transverse relaxation time (T2) in both aqueous suspensions and tissue mimicking phantom gels using MRI. The use of this size separation technique will allow for important size studies to be conducted in the future that will be critical to the optimization of magnetic nanoparticle for biomedical applications.