Histone deacetylase (HDAC) enzymes modulate gene expression through the deacetylation of lysine residues on histone proteins and act as transcriptional repressors of genes. Based on their role in cell cycling, apoptosis and differentiation, HDACs have been chosen as therapeutic targets for the treatment of cancer and neurodegenerative diseases. The SensoLyte® 520 HDAC Activity Assay Kit provides a convenient, two-step homogeneous procedure for measuring HDAC activity using a fluorogenic substrate. In the first step, an acetylated substrate is incubated with HDAC containing samples. Deacetylation of substrate sensitizes it, so that, in the second step mixing with the HDAC developer generates a fluorophore, which can then be detected at Ex/Em= 490 nm/520 nm. The substrate included in the kit is cell-permeable, and the assay can be used to measure HDAC activity directly in cell culture in a 96-well plate without a time-consuming cell extraction step. This kit also can be used for high throughput screening of HDAC inhibitors using extracts or purified enzymes. The long wavelength fluorescence of HDAC 520 substrate is less interfered by the autofluorescence of cell components and test compounds. The kit contains: • HDAC 520 substrate • Deacetylated reference standard for calibration • HeLa nuclear extract • Assay buffer • Cell lysis buffer • HDAC developer • Trichostatin A (HDAC inhibitor) • A detailed protocol.