Abstract Histone deacetylase 6 (HDAC6) is primarily located in the cytoplasm and is responsible for deacetylating non-histone substrates, including α-tubulin, cortactin, and HSP90. HDAC6 possesses a unique structure with two catalytic domains (CD), each containing a catalytic zinc ion, and a zinc finger ubiquitin-binding domain. The structure-based design of HDAC6 inhibitors relies on the use of the zebrafish surrogate. Additionally, there are no reports of selective class IIb inhibitors lacking the zinc-binding group that targets the catalytic zinc ion. We have identified a novel, HDAC6 inhibitor named BAS-2. This inhibitor has a unique structure without an apparent zinc-binding group, and its binding mode is currently unknown. In this study, we employed a combined approach of structure-activity relationships (SAR), molecular modeling, mutational studies, and the application of novel Proteolysis Targeting Chimeras (PROTACs) technology to uncover the binding mode. Systematic variations indicated that BAS-2’s amide group was potentially in a solvent-exposed region. This observation was confirmed by the successful design of a PROTAC that induced HDAC6 degradation in a proteasomal dependent manner. Our findings revealed that BAS-2 did not inhibit the zebrafish surrogate HDAC6 enzyme. Using computational modelling we identified an essential role for aspartate 567 in HDAC6. This was then confirmed by mutational studies in cells, in the binding of BAS-2 to hHDAC6. This discovery opens up opportunities for pioneering the development of the next generation of HDAC6 inhibitors and degraders.