One of the earliest and most common methods for measuring cell viability is the trypan blue (TB) exclusion assay. Trypan blue is a ~960 Daltons molecule that is cell membrane impermeable and therefore only enters cells with compromised membranes. Upon entry into the cell, trypan blue binds to intracellular proteins thereby rendering the cells a bluish color. The trypan blue exclusion assay allows for a direct identification and enumeration of live (unstained) and dead (blue) cells in a given population. Although trypan blue has been used to determine cell viability for many years, it is not without its drawbacks. It is considered to be carcinogenic and must be handled with care and disposed of properly. Over time trypan blue naturally forms dye aggregates and crystals, it is therefore recommended that TB is filtered using a 0.2 micron filter prior to use. Finally, multiple publications have observed that trypan blue viability measurements in samples that are lower than 70 % viable show higher measured cell viability when compared to fluorescent based detection methods 1,2. Data suggests that conducting viability measurements with trypan blue on samples with low viability may not be optimal. We recommend that trypan blue assay is ideally used for cultured cell lines and purified/isolated cell samples with viabilities greater than 70%.