HIV-1 protease (PR) inhibitor (PI) is one of the most potent anti-HIV drugs. When it is used in combination with other drugs, if could suppress HIV to an undetectable level. However, successful treatment is often threaten by emergence of viral drug resistance of PRs (vdrPRs). Three vdrPRs were isolated from HIV-infected patients that carry 7 (M7PR), 10 (M10PR) and 11 (M11PR) PR gene mutations, respectively. They were expressed in a gene-inducible fission yeast system to allow the measurement of PR-specific activities. All 3 vdrPRs proteolyzed natural HIV viral substrates and conferred drug resistance to Indinavir in the fission yeast, suggesting they maintained the same proteolytic and drug resistant activities in the fission yeast as in mammalian cells. Moreover, the viral enzymatic activities of these vdrPRs coupled with the induction of growth inhibition and cell death, which could potentially be used as endpoints to test the efficacy of PI activities. In this study, 5 investigational PIs were used to test the utility of the PR-producing yeast system with Darunavir (DRV) as a control. All 6 compounds suppressed the wildtype PR and the M7PR-mediated activities. However, none of them suppressed activities conferred by M10PR or M11PR. The fact that M10PR and M11PR were resistant to all of the existing PI drugs including DRV, underscores the importance of continued searching for new PIs against vdrPRs. The described fission yeast cell-based system might be suitable for future testing or discovery of new PIs through high-throughput drug screening. Because this yeast cell-based method is function-driven. It has no presumption of what kind of PI will be found. It has the potential to uncover novel PIs.