Allosteric regulation in proteins is fundamental to many important biological processes. Allostery has been employed to control protein functions by regulating protein activity. Engineered allosteric regulation allows controlling protein activity in subsecond time scale and has a broad range of applications, from dissecting spatiotemporal dynamics in biochemical cascades to applications in biotechnology and medicine. Here, we review the concept of allostery in proteins and various approaches to identify allosteric sites and pathways. We then provide an overview of strategies and tools used in allosteric protein regulation and their utility in biological applications. We highlight various classes of proteins, where regulation is achieved through allostery. Finally, we analyze the current problems, critical challenges, and future prospective in achieving allosteric regulation in proteins.