Human respiratory syncytial virus (RSV) encodes multifunctional viral proteins that form interactions with each other and with host proteins during different stages of the viral replication cycle to facilitate viral replication and pathogenesis. Biochemical and biophysical characterization of protein-protein interactions is important for validation and for providing insights into the regulatory mechanisms used by viral proteins during RSV infections. In this chapter, we describe a process that can be used to validate direct protein-protein interactions by in vitro pulldown assay and biophysical characterization using size-exclusion chromatography-multiangle light scattering (SEC-MALS) and hydrogen-deuterium exchange-mass spectrometry (HDX-MS).