Protein kinase CK2 (formerly known as casein kinase 2 or II), a ubiquitous and constitutively active enzyme, is widely recognized as one of the most pleiotropic serine/threonine kinases. It plays a critical role in numerous signaling pathways, with hundreds of bona fide substrates. However, despite considerable research efforts, our understanding of the entire CK2 substratome and its functional associations with the majority of these substrates is far from being completely deciphered. In this context, we aim to provide an overview of how CK2 recognizes its substrates. We will discuss the pros and cons of the existing methods to manipulate CK2 activity in cells, as well as exploring the dynamic response of substrate phosphorylation to CK2 modulation.