Pull-down techniques are essential for identifying direct protein–protein interactions in vitro and play a pivotal role in the study of virus–host protein interactions. These methods rely on a bait protein immobilized on a solid support to capture target prey proteins, which are subsequently eluted and identified. In the context of viral infection research, pull-down techniques offer high specificity and low background interference, which are crucial for elucidating protein interaction networks and investigating cellular signaling pathways. This chapter provides a detailed description of the experimental methods for validating virus–host protein interactions through pull-down techniques, offers vital insights into the molecular mechanisms underlying viral infections, and serves as a cornerstone for drug development and therapeutic strategies.