Histone deacetylases also known as lysine deacetylases (HDACs or KDACs) catalyze the hydrolysis of acyl groups from ε-N-acetyl lysine of protein substrates. Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family due to its complex domain organization and cytosolic localization. HDAC6 exerts its enzymatic activity on many non-histone substrates such as tubulin, heat shock protein 90 (Hsp90), survivin, and cortactin. It has been shown that inhibition of HDAC6 enzymatic activity can be used potentially for the therapy of several neurological conditions including peripheral neuropathies, Alzheimer`s disease, and Parkinson’s disease. Unfortunately, however, currently no truly HDAC6-specific compounds exist to meet clinical needs. We have implemented in our laboratory an innovative screening cascade comprising in vitro isotype specificity testing and in vitro/in vivo toxicity and tubulin biomarker assays. Assisted by our X-ray crystallography data we designed and optimized novel HDAC6 inhibitors bearing an alternative non-hydroxamate zinc-binding group. Such compounds have nanomolar potency and exquisite 10,000-fold selectivity for HDAC6 over other HDAC isoforms and can serve as either research tools or leads for the development of compounds used in the clinical practice.