Nuclear magnetic resonance (NMR) spectroscopy in solution has a unique potential for providing novel insights into structural and dynamic aspects of the solvation of biological macromolecules such as proteins, which is based on the observation of intermolecular solvent-protein nuclear Overhauser effects (NOEs) in the laboratory frame and the rotating frame. In these experiments spectral overlap between resonance lines of the solvent and the macromolecule may make the distinction between certain water-protein and protein-protein NOEs ambiguous even in two- and higher-dimensional NMR experiments. Here we show that use of diffusion filters in NOE difference experiments enables the observation of hydration water molecules without interference from intramolecular NOEs. Water-protein NOEs can thus be unambiguously identified from measurements at a single set of solution conditions. The proposed approach works well also with very short mixing times, and the resulting spectra are easy to analyze. One-dimensional experiments with diffusion filters have been combined with higher-dimensional experiments to obtain the chemical shift dispersion needed for individual resonance assignments.