BACKGROUND: Protein kinases represent one of the most promising groups of drug targets owing to their involvement in such pathological conditions as cancer, inflammatory diseases, neural disorders, and metabolism problems. In the last few years, numerous pharmaceutical and biotech companies have established kinase high-throughput screening (HTS) programs, and the reagent and service industries for kinase assay platforms, kits, and profiling services have begun to thrive. OBJECTIVE: The plethora of different assay formats available today poses a great challenge to scientists who want to select a technology that will be cost efficient, convenient to use, and have low false positive and false negative rates. METHODS: In the current review, we summarize the most commonly used kinase assay methods in the drug discovery process, present the advantages and disadvantages of each of these methods, and discuss the challenges of discovering kinase inhibitors by using these technologies. CONCLUSIONS: The decision of selecting the assay formats for HTS or service platform for profiling should take into account not only the final goals of the screens but also the limitation of resources.