Aberrant protein kinase signaling is a hallmark of many human diseases including cancer, diabetes, and neurological disorders. Kinase inhibitors have shown to be successful at treating some of these diseases, implying that understanding kinase signaling pathways may lead to additional, non-kinase drug targets. However, identifying substrates of protein kinases is difficult due to the universality of the chemical mechanism kinases utilize and the ability of multiple kinases to phosphorylate the same protein substrates. In this review, we explore the advantages and disadvantages of several techniques for identifying kinase substrates. Once putative substrates are identified, their validation as physiological substrates remains a major challenge. We propose three criteria for confirming the physiological relevance of a putative substrate's interaction with a kinase.