A modified HIV gp120 protein labeled with a fluorophore has been used herein in single-molecule imaging techniques to demonstrate the conformation states of HIV-1 Env. The introduction of small organic fluorophores at selected positions within HIV-1 envelope protein gp120 that do not affect infectivity permits the detection of changes in inter-dye distances as a measure of conformational changes. Implementation of the smFRET-based technologies disclosed herein enable conformational screening for molecules that block, induce or trap HIV-1 Env in specific conformational states. Methods for identifying anti-HIV drugs in a smFRET-based screening, and various reagents useful for implementation of such methods, are provided.