Hydrophobicity is an important physicochemical property of peptides in solution. As well as being strongly associated with peptide stability and aggregation, hydrophobicity governs the solution based chromatographic separation processes, specifically reversed-phase liquid chromatography (RPLC). In addition, hydrophobicity is a major physicochemical property of peptides in comparison to H-bonding, electrostatic, and aromatic properties in intermolecular interactions. However, a wide range of molecular factors can influence peptide hydrophobicity, with accurate predictions depending on specific peptide amino acid compositions, structure, and conformation. It is noticeable that peptide composition, the position of the amino acid, and its neighbouring groups play a crucial role in the elution process. In light of this, the same amino acid behaved differently depending on its position and neighbouring amino acid in the peptide chain. Extra attention should be paid to the denaturation process during the course of elution, as it has been shown to complicate and alter the elution pattern. This paper reports on the key peptide properties that can alter hydrophobicity and, consequently, the RPLC elution behaviour of the peptides, and it will conclude by proposing improved prediction algorithms for peptide elution in RPLC.