A cell-based drug screening system that utilizes a green fluorescent protein (GFP)-tagged recombinant lentiviral vector has been used to screen a chemical library of 34,000 small molecules for antiretroviral compounds. Thirty-three initial hits were analyzed and four compounds were selected based on their anti-human immunodeficiency virus type 1 (HIV-1) activity (EC(50) values ranging from 0.17 to 1.9 μM) and low cellular toxicity (CC(50) values >50 μM). The four compounds blocked reverse transcription and were able to inhibit the replication of a panel of different HIV-1 strains, including non-B subtype and viruses resistant to different drug classes. Serial in vitro passages of HIV-1(B-HXB2) in the presence of increasing drug concentrations selected for viruses with reduced susceptibility. Mutations previously associated with resistance to non-nucleoside reverse transcriptase (RT) inhibitors (L100I and Y181C for CBL-17 and CBL-21, respectively) or linked to nucleoside analogue resistance (A62V for CBL-4.0 and CBL-4.1) were identified. Viruses with reduced susceptibility to CBL-17 and CBL-21 but not the ones resistant to CBL-4.0 or CBL-4.1 showed a decrease in replicative fitness. Interestingly, two of the small molecules (CBL-4.0 and CBL-4.1) are indolopyridinones that were previously described as nucleotide-competing RT inhibitors.