Cell sorting involves isolating a uniform population from a group of diverse cells. This method employs advanced techniques like fluorescence-activated cell sorting (FACS) to precisely separate cells based on their unique functional and immune-phenotypic properties. Researchers can then conduct additional experiments and analysis on the separated cells, expanding possibilities for important discoveries in areas such as immunology, infectious diseases, regenerative medicine, and oncology. Cell sorting is now widely used in clinical diagnosis and biomedical research. This comprehensive chapter provides an in-depth exploration of cell sorting procedures, covering the prerequisites, various cell separation techniques, their mechanisms, and the pros and cons. It concludes with a compelling discussion on the application of cell sorting technology.