The effect of histone deacetylases (HDACs) on normal and aberrant gene expression has been studied widely, making these enzymes interesting targets for the treatment of cancer and other diseases. In this chapter, we present in vitro assays that are commonly used to detect HDAC activity that do not rely on radioactive substrates and are amenable for high-throughput testing in microtiter plates. The major focus is on in vitro screening, but we also provide protocols to monitor HDAC activity from cancer cells and peripheral white blood cells. We will discuss the advantages and drawbacks of the respective protocols and give general hints and suggestions that are valuable to obtain reliable and reproducible results.