Hydrophobicity is the association of non-polar groups or molecules in an aqueous environment which arises from the tendency of water to exclude non-polar molecules. Hydrophobic interaction is one of the most important non-bonded interactions which determine protein folding and ligand-receptor binding. To date, hydrophobicity usually has to be evaluated by means of calculation of LogP, because it is a tedious procedure to experimentally measure the hydrophobicity of molecules using techniques reported previously. As a matter of fact, the nature of hydrophobicity in chemistry refers to the physical property of a molecule that is repelled by water, which is clearly associated with the non-polar area in the molecules. The later can be correlated to signal intensity of the molecules in electrospray ionization mass spectrometry. Various dilute water solutions of tripeptides with increasing non-polar area were investigated in ESI time-of-flight mass spectrometry, and the ESI response of the tripeptides was linearly correlated to the non-polar areas in the molecules. Therefore, the hydrophobicity of peptides can be measured quantitatively in terms of ESI signal intensity. Thus hydrophobicity of the peptides was experimentally measured within a few minutes, and data showed a good agreement with previous results obtained in high performance liquid chromatography.