High-throughput screening (HTS) assays for protein-ligand interactions employing mass spectrometric detection are invaluable in early-stage drug discovery. We introduce an advanced Collision-Induced Affinity Selection (CIAS) mass spectrometry (MS) platform, a new addition to the ASMS family, specifically tailored for ligand discovery. Unlike traditional approaches requiring multiple preparation steps, our method eliminates extra steps by integrating all key processes—separation, dissociation, selection, and detection—directly within the mass spectrometer. This streamlined HTS system applies both positive and negative ion modes to ensure comprehensive ligand coverage following dissociation. To further enhance the platform's utility, we establish quantitative binding affinity measurements by calculating the linear slope of released ligands at near-zero concentrations, providing an efficient method for evaluating interaction strengths. Building on previous ligand screening results, the platform was validated using Nsp9, a SARS-CoV-2 protein involved in RNA replication, to assess its ability to accurately reflect binding affinity with known ligands. This mix-and-measure screening approach represents a robust, high-throughput solution for rapid and accurate protein-ligand interaction analysis, offering significant advancements in the field of ligand screening and drug discovery.