Stem-loop 3 RNA (SL3) in ψ-RNA is a highly conserved motif in different strains of HIV-1 and serves as a principle determinant for viral packaging. Viral encapsulation is critical for viral replication, and disruption of the nucleocapsid−ψ-RNA complex interferes with viral replication. We have used SL3 RNA as a target for identification of small molecule inhibitors of the interactions of nucleocapsid protein (NCp7) and ψ-RNA. We report the use of computational and high-throughput screening approaches to identify 16 compounds that bind SL3 RNA with micromolar affinities. Among the identified ligands, two molecules, compounds 7 and 17, bind with higher affinity to SL3 RNA than to double- and single-stranded RNAs. Four of the 16 SL3 RNA ligands inhibit interactions between SL3 RNA and NCp7 with micromolar inhibition constants. In general, the identified SL3 ligands have simple molecular structures and low molecular weights and are, therefore, possible lead compounds for the development of ligands that target the elements of ψ-RNA of HIV-1 with high affinity and specificity.