Fraction unbound () of liver tissue, hepatocytes, and other cell types is an essential parameter used to estimate unbound liver drug concentration and intracellular free drug concentration. and are frequently measured in multiple species and cell types in drug discovery and development for various applications. A comparison study of 12 matrices for and of hepatocytes in five different species (mouse, rat, dog, monkey, and human), as well as of Huh7 and human embryonic kidney 293 cell lines, was conducted for 22 structurally diverse compounds with the equilibrium dialysis method. Using an average bioequivalence approach, our results show that the average difference in binding to liver tissue, hepatocytes, or different cell types was within 2-fold of that of the rat Therefore, we recommend using rat as a surrogate for liver binding in other species and cell types in drug discovery. This strategy offers the potential to simplify binding studies and reduce cost, thereby enabling a more effective and practical determination of for liver tissues, hepatocytes, and other cell types. In addition, under hepatocyte stability incubation conditions should not be confused with , as one is a diluted and the other is an undiluted Cell density also plays a critical role in the accurate measurement of.