Histone deacetylase inhibitors (HDACi) represent a promising avenue for cancer therapy. We applied mass spectrometry (MS) to determine the impact of clinically relevant HDACi on global levels of histone acetylation. Intact histone profiling revealed that the HDACi SAHA and MS-275 globally increased histone H3 and H4 acetylation in both normal diploid fibroblasts and transformed human cells. Histone H3 lysine 56 acetylation (H3K56ac) recently elicited much interest and controversy due to its potential as a diagnostic and prognostic marker for a broad diversity of cancers. Using quantitative MS, we demonstrate that H3K56ac is much less abundant than previously reported in human cells. Unexpectedly, in contrast to H3/H4 N-terminal tail acetylation, H3K56ac did not increase in response to inhibitors of each class of HDACs. In addition, we demonstrate that antibodies raised against H3K56ac peptides cross-react against H3 N-terminal tail acetylation sites that carry sequence similarity to residues flanking H3K56.