CellTrace™ Violet (CTV) is a powerful tool for tracking cell proliferation by permanently binding cellular proteins and rendering the cell fluorescent. After cell division, each daughter cell contains half of the parent cell's fluorescence, enabling quantification of proliferation via flow cytometry. This method enables monitoring of several generations of cell division and tracking of different cell populations in co-culture. Here we describe the use of CellTrace™ Violet in different cell types, and we share important observations we made during protocol optimization.