A novel methodology is shown enabling the screening of mixtures of compounds for their affinity to a receptor protein. The system presented, dynamic protein-affinity chromatography solid-phase extraction (DPAC-SPE), overcomes the limitations of the existing methods by performing an incubation of the His-tagged protein with a mixture of possible ligands, which are still in their native conditions. This is followed by a fully automated affinity trapping step, coupled on-line to an LC-MS system in order to detect and identify the bound ligands. The system has been optimized using a commercially available on-line SPE system, using the estrogen receptor alpha (ERalpha) as model protein. A representative range of ligands with sub-nanomolar to millimolar affinities has been identified successfully from a mixture. The weakest binder that can be identified is norethindrone (approximately K(d)=0.1-1mM). The same setup also provides the possibilities to measure EC50 curves of both weak and strong binders.