Monitoring cell viability is a crucial task essential for the fundamental studies in apoptosis, necrosis, and drug discovery. Cell apoptosis and necrosis are significant to maintain the cell population, and their abnormality can lead to severe diseases including cancer. During cell death, significant changes occur in the intracellular contents and physical properties, such as decrease of esterase activity, depolarization of the mitochondrial membrane potential (ΔΨm), increase of caspase content, dissipation of membrane asymmetry, and loss of membrane integrity. To detect cell viability, the fluorescent probes have been developed by taking advantage of these biological parameters and using various fluorescence mechanisms. These fluorescent probes can serve as powerful tools to facilitate the research in biology and pathology. In this Account, the representative examples of the fluorescent probes for cell viability during the past decades have been summarized and classified into five types based on the biological changes. The basic principle, design strategy, fluorescence mechanisms, and molecular construction of these fluorescent probes have been discussed. Furthermore, the intrinsic characteristics and merits of these probes have been illustrated. Particularly, this Account describes our recent works for the design and synthesis of the fluorescent probes to detect cell viability in the dual-color and reversible modes. The dual-color and reversible fluorescent probes are highlighted owing to their unique benefits in accurate and dynamic detection of cell viability. In general, the dual-color fluorescent probes were constructed based on the loss of esterase activity during cell death. Excited-state intramolecular proton transfer (ESIPT) and intramolecular charge transfer (ICT) process were exploited for the probe design. The construction of such dual-color probes were realized by the acetate of the phenyl group on fluorophores. Esterases in healthy cells hydrolyze the acetate and bring a spectral shift to the probes. Moreover, reversible fluorescent probes for cell viability were designed based on the depolarization of ΔΨm, with relocalization properties dependent on ΔΨm. The probes target mitochondria in healthy cells with high ΔΨm, while they are relocalized into the nucleus in unhealthy cells with depolarized ΔΨm. As ΔΨm is reversibly changed according to the cell viability, these probes reversibly detect cell viability. The reversible and simultaneously dual-color fluorescent probes were developed based on the relocalization mode and aggregation-induced emission shift. The probes target mitochondria to form aggregates with deep-red emission, while they migrate into the nucleus to present in monomers with green fluorescence. In this manner, the probes enable dual-color and reversible detection of cell viability. Fluorescent probes for cell viability based on sensing the membrane integrity, caspase activity, and membrane symmetry are also presented. High-polarity and large-size fluorescent probes impermeable to the intact lipid bilayer selectively target apoptotic cells with a destructive plasma membrane. Fluorescent probes sensing caspases in a turn-on manner exclusively light up apoptotic cells with caspase expression. Membrane-impermeable probes with high affinity to phosphatidylserine (PS) specifically stain the plasma membrane of dead cells, since PS flip-flops to the outer leaflet of the membrane during cell death. In summary, this Account illustrates the basic principles, design strategies, characteristics, and advantages of the fluorescent probes for cell viability, and it highlights the dual-color and reversible probes, which can promote the development of fluorescent probes, apoptosis studies, drug discovery, and other relative areas.