The class IIb histone deacetylase HDAC10 is responsible for the deacetylation of intracellular polyamines, in particular N⁸-acetylspermidine. HDAC10 is emerging as an attractive target for drug design owing to its role as an inducer of autophagy, and high-resolution crystal structures enable structure-based drug design efforts. The only crystal structure available to date is that of HDAC10 from Danio rerio (zebrafish), but a construct containing the A24E and D94A substitutions yields an active site contour that more closely resembles that of human HDAC10. The use of this “humanized” construct has advanced our understanding of HDAC10-inhibitor structure-activity relationships. Here, we outline the preparation, purification, assay, and crystallization of humanized zebrafish HDAC10-inhibitor complexes. The plasmid containing the humanized zebrafish HDAC10 construct for heterologous expression in Escherichia coli is available through Addgene (#225542).