Updated project metadata. Artemetin is a valuable 5-hydroxy-3,6,7,3',4'-pentamethoxyflavone found in many different medicinal plants with a very good oral bioavailability and drug-likeness values endowed with numerous bioactivities such as anti-inflammatory and anti-cancer ones. Here, a multi-disciplinary strategy has been developed and applied for identifying Artemetin target(s) to fully address its mechanism of action, based on drug affinity response target stability and targeted limited proteolysis. Both approaches point to the identification of Filamins A and B as major Artemetin targets in HeLa cell lysates, giving also detailed insights into the ligand/proteins interaction sites. Interestingly, also the 8-prenyl-Artemetin, which is a more permeable semisynthetic analog, directly interacts with Filamin A and B. Both compounds alter filamins conformation in living HeLa cells with an effect on cytoskeleton disassembly and on the disorganization of the F-actin filaments, mediated by the dissociation of filamins. Both the natural compound and its derivative are able to block cell migration, expectantly acting on tumor metastasis’s occurrence and development.