Research interest in the development of histone deacetylase 8 (HDAC8) activators has substantially increased since loss-of-function HDAC8 mutations were found in patients with Cornelia de Lange syndrome (CdLS). A series of N-acetylthioureas (e.g., TM-2-51) have been identified as HDAC8-selective activators, among others; however, their activation mechanisms remain elusive. Herein, we performed molecular dynamics (MD) simulations and fragment-centric topographical mapping (FCTM) to investigate the mechanism of HDAC8 activation. Our results revealed that improper binding of the coumarin group of fluorescent substrates leads to the “flipping out” of catalytic residue Y306, which reduces the enzymatic activity of HDAC8 towards fluorescent substrates. A pocket between the coumarin group of the substrate and thed catalytic residue Y306 was filled with the activator TM-2-51, which not only enhanced binding between HDAC8 and the fluorescent substrate complex but also stabilized Y306 in a catalytically active conformation. Based on this newly proposed substrate-dependent activation mechanism, we performed structure-based virtual screening and successfully identified low-molecular-weight scaffolds as new HDAC8 activators.