Recently there has been renewed interest in Affinity Selection Mass Spectrometry (ASMS) based high throughput screening approaches. Here we describe an approach using the Automated Ligand Identification System (ALIS) to identify test compounds that bind protein or nucleic acid targets of interest from pools of hundreds of small molecule test compounds. The ALIS approach uses 2D size exclusion/reversed phase chromatography coupled to a high resolution time of flight mass spectrometer to identify unique binders in pools of up to 600 test compounds in 5.5 minutes. Data analysis and hit identification is performed using a combination of Agilent Mass Hunter and proprietary custom software. Putative primary screening hits are confirmed in singleton or small pools of up to 5 compounds at 3 different concentrations. Validated hits can be followed up as full dose titrations to determine binding characteristics and affinity. With this approach, high throughput screens with up to 125K test compounds can be completed in less than 36 hours and require only a few mg of target protein or nucleic acid.