Histone deacetylases (HDACs) are key regulators of gene expression that require assembly into larger protein complexes for activity. Efforts to understand how associated proteins modulate the function of HDACs would benefit from new technologies that evaluate HDAC activity in native biological systems. Here, we describe an active site-directed chemical probe for profiling HDACs in native proteomes and live cells. This probe, designated SAHA-BPyne, contains structural elements of the general HDAC inhibitor suberoylanilide hydroxamic acid (SAHA), as well as benzophenone and alkyne moieties to effect covalent modification and enrichment of HDACs, respectively. Both class I and II HDACs were identified as specific targets of SAHA-BPyne in proteomes. Interestingly, multiple HDAC-associated proteins were also enriched by SAHA-BPyne, even after denaturation of probe-labeled proteomes. These data indicate that certain HDAC-associated proteins are directly modified by SAHA-BPyne, placing them in close proximity to HDAC active sites where they would be primed to regulate substrate recognition and activity. We further show that SAHA-BPyne can be used to measure differences in HDAC content and complex assembly in human disease models. This chemical proteomics probe should thus prove valuable for profiling both the activity state of HDACs and the binding proteins that regulate their function.