We will enable an ultra high throughput screening strategy to identify novel potent inhibitors of Human Immunodeficiency Virus (HIV) particle assembly. Current treatments of HIV-infected AIDS patients are based on combinations of drugs that target several critical key steps in the early and late stages of the viral replication cycle. While highly effective drugs have been developed, continuous emergence of multi-resistant viral strains requires the discovery of new classes of drugs directed against novel HIV targets. We will focus on the capsid protein (CA), the main component of the HIV virion: its dimerization is the first step in assembly of the viral particle. No anti-HIV drugs have yet been clinically developed against this target. Methods to measure capsid multimerization have however been recently used to identify proof-of-principle peptides and small compound inhibitors which block viral assembly and HIV replication, including in HIV strains resistant to drugs targeting viral enzymes. We designed an ultra High-Throughput Screening-amenable assay (uHTS), and companion screens and counter-screens to identify potent and selective inhibitors of capsid assembly. By combining transfer-of-energy biochemical screens with secondary assays in HIV-infected cells, we will enable the discovery of novel compounds of high affinity. We will successively i) perform a primary uHTS screen in a 1,536 well microplate format to identify inhibitors of HIV CA dimerization in the well-precedented 350,000 compound MPLCN library ii) triage compounds interfering non-specifically with the specific or unrelated protein-protein assays, and eliminate hits cytotoxic for cells used for studying effects on HIV, ii) evaluate hit compounds on HIV-infected cells for their ability to inhibit CA production and HIV infectivity, iv) verify the stage of the life cycle affected by the inhibitors, v) survey the spectum of HIV-1 and HIV-2 laboratory-adapted and clinical strains inhibited by our compounds. To demonstrate dimerization using alternately tagged CA proteins, we designed a set of complementary and orthogonal assays. The primary TR-FRET assay will be optimized in 384-well microtiter plates and validated in pilot screens of model Libraries (e.g.: LOPAC) before running a large MPLCN screen, using as references the NYAD-1 peptide and the PF-74 small compound capsid inhibitors. Our HIV CA-specific AlphaScreen will serve as a companion assay to help characterize hit compounds for further biologic validation. Several counter-screens will serve to eliminate false-positive hits. CA-specific inhibitors will be differentiated by their capaity to inhibit interactions between the N-terminal and C-terminal portions of the CA protein, by using CA-based assays. Secondary cell-based assays HIV infectivity assays will be used to characterize non cytotoxic specific high-affinity inhibitors of HIV replication. The stage of the HV replication cycle targeted by the hits will be identified in comparison with other known anti-HIV compounds including AZT, Efavirenz and Saquinavir. Our goal is to provide the scientific community with freely accessible robust assays and potent, diversely acting HIV capsid inhibitors active in nanomolar concentrations.