The application of surface plasmon resonance (SPR)-based optical biosensors has contributed extensively to our understanding of functional aspects of HIV. SPR biosensors allow the analysis of real-time interactions of any biomolecule, be it protein, nucleic acid, lipid, carbohydrate or small molecule, without the need for intrinsic or extrinsic probes. As such, the technology has been used to analyze molecular interactions associated with every aspect of the viral life cycle, from basic studies of binding events occurring during docking, replication, budding and maturation to applied research related to vaccine and inhibitory drug development. Along the way, SPR biosensors have provided a unique and detailed view into the inner workings of HIV.