Histone deacetylase 10 (HDAC10) is unique among the greater HDAC family due to its unusually narrow substrate specificity as a polyamine deacetylase, specifically as an N ⁸-acetylspermidine hydrolase. Polyamines are essential for cell growth and proliferation; consequently, inhibition of polyamine deacetylation represents a possible strategy for cancer chemotherapy. In this work, we have designed six acetylated phenylthioketone inhibitors of HDAC10 containing positively charged para– and meta-substituted amino groups designed to target interactions with E274, the gatekeeper that recognizes the positively charged ammonium group of the substrate N ⁸-acetylspermidine. We prepared each of these inhibitors through a short synthetic route of six steps. By adapting a low-cost colorimetric activity assay, we measured low-micromolar IC₅₀ values for these compounds against a humanized construct of zebrafish HDAC10 (A24E-D94A HDAC10). Selected inhibitors were cocrystallized with A24E-D94A zebrafish HDAC10 and zebrafish HDAC6 to provide insight into class IIb isozyme affinity and selectivity.