High-throughput screening of combinatorial libraries has evolved from studying large diverse libraries to analyzing small, structurally similar, focused libraries. This paradigm shift has generated a need for rapid screening technologies to screen both diverse and focused libraries in a simple, efficient, and inexpensive manner. We have proactively addressed these needs by developing a high-throughput, solution-based method combining size exclusion (SEC), two-dimensional liquid chromatography (2-D LC), and mass spectrometry (MS) for determining the relative binding of drug candidates in small, focused medicinal libraries against human serum albumin (HSA). Two types of libraries were used to evaluate the performance of the system. The first consisted of five diverse ligands with a wide range of hydrophobicities and whose association constants to HSA cover 3 orders of magnitude. A beta-lactam library composed of structurally similar compounds was used to further confirm the validity of the methodology. The ability to distinguish site-specific interactions of drugs competing for individual domains of the HSA receptor is also demonstrated. Comparison of chromatographic profiles of the library components before and after incubation with the receptor using multiple reaction monitoring allowed a ranking of the ligands according to their relative binding affinities. The observed rankings correlate closely with literature values of the association constants between the respective ligands and HSA. This simple, rugged methodology can screen a wide spectrum of chemical entities from combinatorial mixtures in less than 6 min.