Abstract Background Inhibitors of the HIV-1 Protease currently used in therapeutic protocols, have been found to inhibit, although at higher concentrations, the HIV-2 encoded enzyme homologue. Similar to observations in HIV-1 infected individuals, therapeutic failure has also been observed for some patients infected with HIV-2 as a consequence of the emergence of viral strains resistant to the anti-retroviral molecules. In order to be able to define the specific mutations in the Protease that confer loss of susceptibility to Protease Inhibitors, we set up an experimental model system based in the expression of the viral protein in yeast. Results Our results show that the HIV-2 Protease activity kills the yeast cell, and this process can be abolished by inhibiting the viral enzyme activity. Since this inhibition is dose dependent, IC₅₀ values can be assessed for each anti-retroviral molecule tested. We then defined the susceptibility of HIV-2 Proteases to Protease Inhibitors by comparing the IC₅₀ values of Proteases from 7 infected individuals to those of a sensitive wild type laboratory adapted strain. Conclusion This functional assay allowed us to show for the first time that the L90M substitution, present in a primary HIV-2 isolate, modifies the HIV-2 Protease susceptibility to Saquinavir but not Lopinavir. Developing a strategy based on the proposed yeast expressing system will contribute to define amino acid substitutions conferring HIV-2 Protease resistance.