Hoechst side population (SP) analysis remains a critical technique for identifying stem cell and progenitor populations in hematopoietic and non-hematopoietic tissues, as well as potential cancer stem cells. More recently, DyeCycle Violet (DCV), a DNA binding dye structurally similar to Hoechst 33342 but with an excitation spectrum shifted toward the violet range, has also been used for SP analysis on flow cytometers equipped with violet laser diodes. In this chapter, we briefly review the history of this method and provide a detailed procedure. Critical parameters for good labeling, details on integrating simultaneous immunolabeling with DCV SP analysis, and proper data acquisition and analysis techniques are covered in detail.