Frontal affinity chromatography (FAC) is a biophysical method for the discovery and characterization of molecular interactions in a flow-based system. Several different modes of analysis are possible by interfacing to the mass spectrometer, including robust single-compound characterizations as well as high-throughput screening of over 1,000 compounds per run. The method supports thermodynamic and kinetic characterization of interactions for a wide range of molecular species and possesses similarities to flow-based biosensors such as surface plasmon resonance. It offers sensitive detection of ligands present well below their respective dissociation constants, and can be assembled from readily available laboratory components. Direct coupling of the FAC cartridge to the mass spectrometer is useful for the interrogation of single compounds or mixtures of limited complexity. An offline fractionation schema is more appropriate for discovery-mode applications. A high-performance FAC system enabling both modes can be assembled in 2-3 h. Measurements of dissociation constants can be made with such a system in 0.5-3 h, and the system supports higher-throughput screening modes at a rate of 10,000 compounds d(-1).