Bio-layer interferometry (BLI) real-time, label-free technology has greatly contributed to advances in vaccine research and development. BLI Octet platforms offer high-throughput, ease of use, reliability, and high precision analysis when compared with common labeling techniques. Many different strategies have been used to immobilize the pathogen or host molecules on BLI biosensors for real-time kinetics and affinity analysis, quantification, or high-throughput titer. These strategies can be used in multiple applications and shed light onto the structural and functional aspects molecules play during pathogen-host interactions. They also provide crucial information on how to achieve protection. This review summarizes some key BLI strategies used in human vaccine research and development.