© 2015 American Chemical Society. Viral assembly, similar to other self-organizing protein systems, relies upon early building blocks, which associate into the late supramolecular structures. An initial and crucial event during HIV-1 core assembly is the dimerization of the capsid protein C-terminal domain, which stabilizes the viral capsid lattice. Thus, monitoring and manipulating this stage is desirable both from mechanistic as well as clinical perspectives. Here, we developed a fluorescent-based method for the detection and visualization of these early capsid interactions. We detected strong dimeric interactions, which were influenced by mutations in the capsid protein. We utilized this assay for potential assembly inhibitors screening, which resulted in the identification of a leading compound that hinders the assembly of capsid protein in vitro. Moreover, a derivative of the compound impaired virus production and infectivity in cell cultures. These findings demonstrate that the described assay efficiently detects the very first association events in HIV-1 capsid formation and emphasize the significance of targeting early intermolecular interactions.