Mutations that occur in response to the HIV-1 protease inhibitors (PIs) are responsible for the development of multi-drug cross-resistance to PIs in AIDS treatment. Virtually all PIs act through the same mechanism: they are transition-state analogs that target the active site of the homodimeric enzyme located at the junction of the two monomers. The emergence of resistance to one PI usually results in cross-resistance to other PIs. One alternative to inhibiting the active site of HIV-1 protease is to target the dimer interface of the enzyme at the antiparallel beta-sheet formed by the interdigitation of the C- and N-ends of each monomer. This region is highly conserved and is responsible for about 75% of the dimer stabilization energy. Here we describe new dimerization inhibitors in which new structural molecular variations have been introduced and the peptidic characteristics have been decreased by introducing peptidomimetic groups that have peptide-like hydrogen bonding properties. This led to an increase of the in vitro efficiency (subnanomolar level) against HIV-1 protease activity. Our dimerization inhibitors proved equally active in vitro against both wild-type and mutated proteases. The mechanism of inhibition was established using a combination of kinetic and biophysical methods. Using analytical ultracentrifugation and NMR, we obtained direct experimental evidence of non-covalent dissociative mode of interaction of the HIV-1 protease dimerization inhibitors