HIV-1 protease is a very attractive target for the development of new anti-HIV drugs and has been extensively studied over the past decades. In this study, we present a detailed atomic level characterization of the dimer interface in the enzyme HIV-1 protease through computational alanine scanning mutagenesis and molecular dynamics simulations. In addition to a full mapping of the amino acid residues present at the subunit interface, in terms of the corresponding energetic contribution for dimer formation and of their dassification as hot spots, warm spots, and null spots, we trace a dynamic analysis of the subunit interacting and solvent accessible surface areas and of the most important hydrogen bonds between subunits. The results presented illustrate the high energetic importance for dimer formation of a small set of five amino acid residue pairs at the subunit interface-LeuS, Ile50, Arg87, Leu97, and Phe99-and provide important dues on the most important structural and energetic determinants for dimer formation. In addition, the results presented suggest several key targets at the subunit interface for the development of new molecules that aim to inhibit HIV-1 protease (PR) activity through blocking the formation of the fully active PR homodimeric form, providing important clues for drug design.