Viral replication is intricately linked to the interaction between viruses and host proteins, making the investigation of host–viral protein complexes vital to virological research. Recent advances in this field have been based on the development of proteomic assays. In this chapter, we discuss the principles, applications, comparative merits, and drawbacks of the most commonly used proteomic methodologies, including co-immunoprecipitation (co-IP), affinity purification–mass spectrometry (AP-MS), liquid chromatography–tandem mass spectrometry (LC-MS), and stable isotope labeling by amino acids in cell culture (SILAC). The Co-IP technique is an in vitro protein–protein interaction assay that has been widely utilized to validate the existence of endogenous protein–protein complexes despite potential issues such as nonspecific binding. AP-MS mainly facilitates purifying and characterizing protein complexes, thereby facilitating the construction of protein interaction networks. LC-MS/MS is utilized to quantify small molecules, which are classified into labeled and unlabeled formats. SILAC can be used to assess proteomic changes dynamically. These techniques, either individually or in combination, provide a comprehensive strategy to experimentally validate and profile the binding affinity of target proteins in host–virus interactions. Comprehending their characteristics and limitations is essential for assessing precise proteomic interaction networks.