Histone deacetylases (HDACs) are ubiquitous enzymes that remove ε-N-acetyl-lysine posttranslational modifications (PTMs) on histone tails. The resulting PTM landscape affects chromatin packing and recruitment of transcription factors, in turn playing an indirect role in regulation of gene expression. Deregulation of the activity of these hydrolases has been associated with several complex diseases. Thus, HDAC inhibitors have been approved for cancer treatment and are being studied against inflammation, neurodegeneration, and autoimmune disorders among others. The role of each of the 11 Zn²⁺-dependent HDACs has not yet been elucidated, mainly due to their structural similarity and, in part, due to the absence of isotype-selective probes. Such selectivity may be achievable by targeting features outside of the active site pocket, which is highly conserved. Peptides, which may cover larger areas than small molecules, may become useful chemical tools able to reach unexplored areas of the protein surface to achieve selectivity. In addition, by incorporating hydroxamic acid-containing lysine mimics in their structure, strong binding to the catalytic cavity is achieved. Furthermore, such molecules present similarities to the native substrates, which could be exploited for determining targets of their deacetylase activity. Therefore, hydroxamic acid-containing peptides have potential for investigating HDAC function. Several examples of the application of these chemotypes are discussed in this book chapter.