Histone deacetylases reside among the most important and novel target classes in oncology. Selective lead structures are intensively developed to improve efficacy and reduce adverse effects. The common assays used so far to identify new lead structures suffer from many false positive hits due to auto-fluorescence of compounds or triggering undesired signal transduction pathways. These drawbacks are eliminated by the dual parameter competition assay reported in this study. The assay involves a new fluorescent inhibitor probe that shows an increase in both, fluorescence anisotropy and fluorescence lifetime upon binding to the enzyme. The assay is well suited for high-throughput screening.