Mass spectrometry-based chemical proteomics enables unbiased assessment of ligand potency and selectivity across the proteome. However, current approaches remain limited by the low throughput of single-compound screening and reliance on pre-synthesized libraries. Here we devise a mechanism-driven “library-versus-proteome” platform that couples dynamic combinatorial libraries with activity-based protein profiling, enabling real-time selection and optimization of ligands in complex biological systems. This approach increases screening throughput by 10- to 20-fold, streamlines library generation, and adopts a “screen first, synthesize later” paradigm. Applying this platform, we discover covalent inhibitors of serine hydrolases including PPME1, ABHD11 and PNPLA6, and reveal uncharacterized roles of PNPLA6 in lipid metabolism and cancer cell proliferation. We further extend the strategy to cysteine-targeting ligands by designing tailored warheads, enabling proteome-wide EC50 profiling of over 2600 ligandable cysteines and yielding inhibitors for NIT2, PRDX5, TXNDC17 and VCP. Focusing on VCP, we uncover a previously unrecognized signaling axis in which GPCR activity modulates activation of the ER stress-induced unfolded protein response. Using a gelbased “library-versus-proteome” assay, we screen over 800 analogues within two days and identify a more potent VCP ligand with nanomolar activity and in vivo antitumor efficacy. This work establishes library-versus-proteome screening as a scalable strategy for ligand discovery.