The MS/NMR Assay is a protocol for rapidly screening small organic molecules for their ability to bind a target protein while obtaining structure related information as part of a structure based drug discovery and design program. The methodology takes advantage of and combines the inherent strengths of size-exclusion gel chromatography, mass spectrometry and NMR to identify bound complexes in a relatively universal high-throughput screening approach. Size-exclusion gel chromatography in the spin column format provides the high-speed separation of a protein-ligand complex from free ligands. The spin column eluent is then analyzed under denaturing conditions by electrospray ionization (ESI) mass spectrometry (MS) for the presence of small molecular-weight compounds formerly bound to the protein. Hits identified by MS are then individually assayed by chemical shift perturbations in a 2D ¹H-¹⁵N-HSQC NMR spectrum to verify specific interactions of the compound with the protein and identification of the binding site on the protein.