As most drugs exert pharmacological effects by interacting with their target proteins, identification of which is a critical step in unravelling the mechanisms of drug action. It is also imperative for our understanding of the pharmacodynamics of a known drug, suggesting the potentially unrevealed actions and thus refining its future clinical applications. However, current in vitro affinity chromatography-based and in vivo activity-based protein profiling (ABPP) approaches generally face difficulties discriminating specific drug targets from non-specific ones. We have come up with a novel approach combining isobaric tags for relative and absolute quantitation (iTRAQTM) with Clickable ABPP, named ICABPP, to specifically and comprehensively identify drug targets in live cells. This approach was applied to identify the protein targets of andrographolide (Andro), a natural product with known anti-inflammation and anti-cancer effects, in live cancer cells. A spectrum of specific targets of Andro was identified, revealing the mechanism of action of the drug and its potential novel application as the tumor metastasis inhibitor, which was validated through cell migration and invasion assays. Moreover, the target binding mechanism of Andro was unveiled with a combination of drug analogue synthesis, protein engineering and mass spectrometry-based approaches and the drug-binding sites of two protein targets, NF-kB and actin, were determined.