We present an extension and confirmation of our previously published method (J. Med. Chem. 2002, 45, 2867-2876) for the prediction of volume of distribution (VD) in humans for neutral and basic compounds. It is based on two experimentally determined physicochemical parameters, ElogD(7.4) and f(i(7.4)), the latter being the fraction of compound ionized at pH 7.4, and on the fraction of free drug in plasma (fu). By regressing the fraction unbound in tissues, fut, vs the above parameters, we demonstrate the ruggedness of the method in predicting VD through the Oie-Tozer equation, via the use of several testing approaches. A comparison is also presented between several methods based on animal pharmacokinetic data, using the same set of proprietary compounds, and it lends further support for the use of this method, as opposed to methods that require the gathering of pharmacokinetic data in laboratory animals. The reduction in the use of animals and the overall faster and cheaper accessibility of the parameters used make this method highly attractive for prospectively predicting the VD of new chemical entities in humans.