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Small molecule pull-down is an affinity purification-based chemical proteomics technique. A biotin or other affinity tag is chemically conjugated to a biologically active small molecule compound to construct a small molecule affinity probe. After incubation with cell lysates or live cells, the probe-target protein complex is specifically captured using streptavidin magnetic beads or agarose beads. Following washing to remove non-specifically bound proteins, the enriched proteins are subjected to Western Blot for targeted validation or high-resolution mass spectrometry for proteomic identification, enabling systematic screening and identification of direct target proteins and their interaction networks. This technique is a core tool for drug target discovery, mechanism of action elucidation, and off-target effect assessment.