HIV disease became a manageable chronic disease since combination antiretroviral therapy (cART) was introduced as the standard treatment regimen. However, the emergence of drug-resistant viruses is a major problem associated with cART. A phenotypic drug susceptibility test using a lentiviral vector was established and applied to evaluate new protease inhibitors (PIs). Lentiviral vectors representing a wild-type (WT-lentivector) and darunavir (DRV)-resistant HIV type 1 (HIV-1) (DRV r-lentivector) were generated. Nine clinically approved protease inhibitors (PIs) inhibited the transduction ability of WT-lentivector similar to their inhibitory effects on the replication of WT HIV-1. Three new PIs reduced the transduction ability of WT- and DRV r-lentivector, suggesting that these PIs may be the candidates as novel antiretroviral drugs against drug-resistant variants of HIV-1.