Infrared spectroscopy identifies molecules by detection of vibrational patterns characteristic of molecular bonds. We apply this approach to measure protein acetylation after treatment with histone deacetylase inhibitors. The anticancer activity of histone deacetylase inhibitors (HDACi) is ascribed to the hyperacetylation of both core nucleosomal histones and nonhistone proteins critical to the maintenance of the malignant phenotype (Marks, P. A.; Richon, V. M.; Breslow, R.; Rifkind, R. A. Curr. Opin. Oncol. 2001, 13, 477-483; Mai, A.; Massa, S.; Rotili, D.; Cerbara, I.; Valente, S.; Pezzi, R.; Simeoni, S.; Ragno, R. Med. Res. Rev. 2005, 25, 261-309). After incubation of the peripheral blood mononuclear cells (PBMCs) in vitro with the HDACi SNDX-275, a benzamide drug derivative, vibrational spectral changes in the methyl and methylene stretching mode regions, which reflect concentration-dependent increases in protein acetylation, were detected and quantified. We applied these metrics, based upon spectral differences, to peripheral blood mononuclear cells from patients treated in vivo with this agent. The data demonstrate a new approach to a sensitive assessment of global molecular modifications that is independent of antibodies, requires minimal cell processing, and is easily adapted to high-throughput screening.