Hydrophobicity is the main determinant in the partitioning of proteins in Aqueous Two-Phase Systems (ATPSs). There are a range of different methods available to evaluate hydrophobicity, including hydrophobic interaction chromatography, reverse phase chromatography, precipitation with ammonium sulphate, average surface hydrophobicity (3-D structure), aminoacid (AA) composition, hydrophobic imbalance, and a statistical description of the AA distribution. This paper reviews the present state of the art in the practical and theoretical evaluation and measurement of a protein's hydrophobicity and evaluates which are the best methodologies to estimate hydrophobicity to predict behaviour in ATPSs. The two methods that are more attractive, depending on the ATPS used, are those that consider the 3-D structure and the hydrophobicity of AA on the surface and the one that uses the parameter 1/m*, which is very simple to determine experimentally.