Objective: To delineate specificity of binding in protein kinases with its substrate or inhibitors so that the design of its inhibitors can be facilitated.Background: For the treatment of a variety of disorders in humans such as cancer, inflammation, diabetes, infections, cardiovascular diseases, etc., protein kinases have been found as a very prominent target and thus attempts have been made to find the potent inhibitors of these kinases. The knowledge of specificity of binding of any enzyme with its substrate or inhibitors greatly facilitates the design of its inhibitors. In this article, thus, attempt has been made to highlight the specific features of kinases that are important for them to bind with the substrates or inhibitors. Method: It is a review article so attempt has been made to compile all the theoretical and experimental studies on specificity of binding in protein kinases and critically analyze them in the light of drugs designed based on such knowledge. Results: From various studies, it has been suggested that all protein kinases have a similar protein fold comprised of two lobes, where one lobe is a small N-lobe made of β -sheets and the other one is a larger C-lobe consisting of α -helices. This protein fold forms an ATP binding cleft that constitutes the active site of the enzyme. Further, the specific features of active site of kinases, that have been found to be usually responsible for its binding preference for the substrate or inhibitors, are its depth, charge, and hydrophobic nature. Conclusion: It has been pictured out that in all protein kinases, the catalytic domain has a hinge region, a hydrophobic channel, a hydrophobic back-pocket and a sugar pocket, which can easily accommodate a substrate or an inhibitor that may complement these features. Protein kinases have also been found to achieve substrate specificity through localization to distinct subcellular compartments or structures. Selectivity of inhibitors also depends on potency. In protein kinases, there is a strong correlation between inhibitor potency and selectivity. Application: This knowledge can be greatly exploited to design and develop potent kinase inhibitors for the treatment of a variety of disorders in humans such as cancer, inflammation, diabetes, infections, cardiovascular diseases, etc.