Changes at the molecular level always occur at different stages in diseased cells. The detection of these changes is critical for understanding the molecular mechanisms underlying pathogenesis, as well as accurately diagnosing disease states and monitoring therapeutic modalities. Cell-SELEX is a foundational tool used to select probes able to recognize molecular signatures on the surface of diseased cells. This technology has been increasingly used in biomarker discovery, as well as cancer diagnosis and therapy. In this chapter, the whole cell-SELEX process is described, including aptamer selection, identification, and validation. In addition, we will explore the challenges and prospects for cell-SELEX now and in the coming years. It is anticipated that this chapter will guide readers toward a better understanding of the working principles underlying the cell-SELEX technology and serve as a practical reference for bench scientists engaged in cell and molecular biology.