<p>Screening of ligands from natural products, especially traditional herbs, has always been an important pathway for drug discovery. In the current work, we present a strategy for the screening of active substances from TCM by combination of in silico target prediction, surface plasmon resonance (SPR), and UPLC-HR-MS/MS, using <i>Kansui</i> as an illustration. In the first, in silico target prediction of diterpenoids from <i>Kansui</i> was performed, aiming to improve the success rate of screening. Carbonic anhydrase II (CA II) were within the most likely targets. Then, SPR combined with UPLC-HR-MS/MS analysis was employed for the screening of ligands for CA II from a mixture of <i>Kansui</i> extract and for the structural identification of the captured ingredients. It was found that the ligands of CA II are mainly ingenane-type diterpenoids, which was further validated by enzymatic activity experiments and molecular docking that ingenane-type diterpenoids have more affinity and potency for CA II activation. It is the first time that the ligands of CA II have been screened from the extract of <i>K</i>ansui, and the present ligand screening work also providing clues to the pharmacological mechanisms and rationality of vinegar-processed <i>Kansui</i>.</p> Graphical Abstract <p></p>