Protein–protein interactions (PPI) are crucial for the establishment of life. However, its basic principles are still elusive and the recognition process is yet to be understood. It is important to look at the biomolecular structural space as a whole, in order to understand the principles behind conformation–function relationships. Since the application of an alanine scanning mutagenesis (ASM) study to the growth hormone it was demonstrated that only a small subset of residues at a protein–protein interface is essential for binding — the hot-spots (HS). Aromatic residues are some of the most typical HS at a protein–protein interface. To investigate the structural role of the interfacial aromatic residues in protein–protein interactions, we performed Molecular Dynamic (MD) simulations of protein–protein complexes in a water environment and calculated a variety of physical–chemical characteristics. ASM studies of single residues and of dimers or high-order clusters were performed to check for cooperativity within aromatic residues. Major differences were found between the behavior of non-HS aromatic residues and HS aromatic residues that can be used to design drugs to block the critical interactions or to predict major interactions at protein–protein complexes.