Next, the selective binding of 5b to Hsp90 was validated in a variety of biochemical and cellular assays, including DARTS, thermal and isothermal shift assays, whereas intracellular Hsp90 engagement and disruption of Hsp90 multiprotein complexes were established via CETSA and ITDRF CETSA assays, and immunoblotting under reducing conditions (+/-DTT) and non-denaturing (BN-PAGE) conditions. Moreover, a crosslinker and auto display dimerization assay, as well as SEC-SAXS measurements, repeatedly confirmed the destabilization of Hsp90α CTD dimers upon 5b incubation, whereas no unspecific binding of 5b was reported on the Hsp90α NTD protein in an FP-based competitive assay.