High-content imaging with robotic microscopy has been widely used for phenotype-based cellular screening research. This technology is ideally suited to monitor intracellular translocation of macromolecules. Here, we describe in detail the procedures for screening microbial natural extracts for their capability to inhibit the general nuclear export machinery. The method is based on human cells that stably express a fluorescent-tagged reporter protein that contain a nuclear export signal capable of mediating its translocation from the nucleus to the cytoplasm through the nuclear pore. In the presence of a small molecule nuclear export inhibitor, the fluorescent signal is trapped to varying degrees within the nucleus. In order to analyze complex libraries of compounds, the assay has been scaled to 96- or 384-well formats and optimized for high-throughput screening (HTS). Active microbial extracts undergo confirmation screening, bioassay-guided fractionation, chemical dereplication, and compound purification. The active purified compound is characterized in secondary assays that monitor the nuclear export of disease-relevant proteins. Nuclear export inhibitors hold promise as potential cancer and antiviral drugs.