Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genomemining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exerttheir function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenouscomplexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, islaborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thusturned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of activeRpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then beused for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay canbe completed within two days. The video component of this article can be found at https://www.jove.com/video/59527/