Due to an involvement in various patho-physiological conditions, human histone deacetylases (HDACs) are high priority drug targets for the treatment of several diseases, such as cancer, heart failure, neurodegeneration, etc. An effector (inhibitor/activator) of these enzymes has a great potential to alleviate the above disease conditions. In this regard, HDAC inhibitors – Zolinza and Istodax - have already been approved by the FDA for the treatment of T-cell lymphoma, aside from several other inhibitors which are in the advanced level of clinical trials. HDAC8 serves as a prototype to study structural-functional and catalytic features of human HDACs. In order to pursue the biophysical studies of the ligand-binding, HDAC8 was cloned, expressed, and purified from E. coli. A high-throughput screening (HTS) of an in-house library of small molecules was performed utilizing a trypsin-coupled in vitro HDAC8 assay to discover novel effectors of HDAC8, and the N-acetylthiourea and the thiopyridine derivatives were discovered as the isozyme-selective inhibitors and activators of HDAC8, respectively. In vitro HDAC8 assay utilizing a fluorogenic peptide as a substrate often produces artifactual results. Therefore, a substrate-independent HDAC8 assay was developed utilizing a fluorescent analog of a pan-HDAC-inhibitor. In view of the fact that the downstream cellular response of a drug is often dictated by the transient kinetic and thermodynamic parameters of its interaction with the target, the transient kinetics and thermodynamics of interaction of the selected HDAC8 inhibitor with the enzyme were thoroughly investigated. It was observed that the dissociation off-rate and/or the enthalpy of binding of an HDAC8 inhibitor to the enzyme could play a crucial role in determining its in vivo efficacy. A rationale has been presented that the above parameters of the ligand-protein interaction could be utilized for optimizing a drug candidate (HDAC8 inhibitor) in order to enhance its in vivo potency.