Histone deacetylases (HDACs) are key regulators of numerous cellular proteins by removing acetylation marks from modified lysine residues. Peptide‐based HDAC probes containing α‐aminosuberic acid ω‐hydroxamate have been established as useful tools for investigating substrate selectivity and composition of endogenous HDAC complexes in cellular lysates. Here we report a structure–activity study of potential HDAC‐probes containing derivatives of the hydroxamate moieties. While most of these probes did not recruit significant amounts of endogenous HDACs from cellular lysates, peptides containing Nε‐acetyl‐Nε‐hydroxy‐L‐lysine served as HDAC probe. The recruitment efficiency varied between HDACs and was generally lower than that of α‐aminosuberic acid ω‐hydroxamate probes, but showed a similar global interaction profile. These findings indicate that Nε‐acetyl‐Nε‐hydroxy‐L‐lysine might be a useful tool for investigations on HDAC complexes and the development of HDAC inhibitors. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.