This manuscript describes an affinity selection-mass spectrometry (AS-MS) method for quantitative protein-ligand binding affinity (Kd) measurements in large compound libraries. The ability of a titrant ligand to displace a target-bound library member-as measured by MS-reveals the affinity ranking of the mixture component relative to "internal affinity calibrants", compounds of known affinity for the target. This technique does not require that the precise concentration of each ligand is known; therefore, unpurified products of mixture-based combinatorial synthesis may be used for affinity optimization and developing structure-activity relationships. The method is demonstrated for a series of ligands to the important oncology target CDK2 that were discovered by AS-MS screening of combinatorial libraries against the basal form of the protein. AS-MS displacement curves for select hits were acquired over a range of compound concentrations, confirming that binding affinity measurement results are concentration-insensitive. These hits were evaluated in pools of purified compounds to verify the method's applicability to hit triage in large chemical libraries. The method was further tested using unpurified, mixture-based combinatorial libraries of >1000 compounds, yielding results that mirror those obtained from mixtures of purified compounds. The technique was then used to identify optimized CDK2 ligands from compound mixtures, quantitatively measure their affinities, and establish structure-activity relationships among these drug leads.