LIM domain kinases 1 and 2 (LIMK1 and LIMK2) regulate actin dynamics and subsequently key cellular functions such as proliferation and migration. LIMK1 and LIMK2 phosphorylate and inactivate cofilin leading to increased actin polymerization. As a result, LIMK inhibitors are emerging as a promising treatment strategy for certain cancers and neurological disorders. High-quality chemical probes are required if the role of these kinases in health and disease is to be understood. To that end, we report the results of a comparative assessment of 17 reported LIMK1/2 inhibitors in a variety of in vitro enzymatic and cellular assays. Our evaluation has identified three compounds (TH-257, LIJTF500025, and LIMKi3) as potent and selective inhibitors suitable for use as in vitro and in vivo pharmacological tools for the study of LIMK function in cell biology.