Of the different hydroxamate-based histone deacetylase (HDAC) inhibitors, suberoylanilide hydroxamic acid (SAHA) has been approved by the Food and Drug Administration for the treatment of T-cell lymphoma. Interestingly, a structurally similar inhibitor, trichostatin A (TSA), which has a higher in vitro inhibitory potency against HDAC8, reportedly shows poor efficacy in clinical settings. To gain molecular insight into this discriminatory feature, we performed transient kinetic and isothermal titration calorimetric studies for the interaction of SAHA and TSA with the recombinant form of human HDAC8. The transient kinetic data revealed that the binding of both inhibitors to the enzyme showed biphasic profiles, which represented an initial encounter of the enzyme with the inhibitor followed by the isomerization of the transient enzyme-inhibitor complexes. The temperature-dependent transient kinetic studies with these inhibitors revealed that the bimolecular process is primarily dominated by favorable enthalpic changes, as opposed to the isomerization step, which is solely contributed by entropic changes. The standard binding enthalpy (ΔH°) of SAHA, deduced from the transient kinetic as well as the isothermal titration calorimetric experiments, was 2-3 kcal/mol higher than that of TSA. The experimental data presented herein suggest that SAHA serves as a preferential (target-specific and -selective) HDAC8 inhibitor as compared to TSA. Arguments that the detailed kinetic and thermodynamic studies may guide the rational design of HDAC inhibitors as therapeutic agents are presented.