Abstract: Polo-like kinases (PLKs) are key enzymes that control mitotic entry of proliferating cells and regulate many aspects of mitosis necessary for successful cytokinesis. Of the four known human PLKs, PLK1 is the best characterized and is overexpressed in many tumour types with aberrant elevation frequently constituting a prognostic indicator of poor disease outcome. Despite the fact that PLK1 has been regarded as a validated mitotic cancer target for a number of years, very few reports of small-molecule PLK inhibitors have appeared to date. In order to provide a starting point for the discovery and development of selective PLK inhibitors, we have characterized a number of known generic kinase inhibitors with hitherto unknown activity against PLK1, as well as discovering novel inhibitors through structure-guided design. Previously, the only characterized biochemical PLK1 inhibitor was scytonemin, a symmetric indolic marine natural product that is a micromolar non-specific ATP competitor. In addition to the progress in the development of ATPcompetitive small-molecule PLK inhibitors, recent reports on the use of antisense oligonucleotides (ASONs) and small interfering RNAs (siRNAs) directed against PLK1 have shown selective antiproliferative effects on cancer cells both in vitro and in vivo, producing phenotypes consistent with known PLK functions, and confirming that targeting PLKs with conventional small-molecule agents may be a valid and effective anticancer strategy. Here we present a progress update on the approaches taken so far in developing PLK inhibitors.