In recent years, numerous membraneless condensates have been found to regulate cellular and viral processes. These condensates are mainly composed of specific proteins with liquid-liquid phase separation (LLPS) properties. Many studies have revealed that these membraneless organelles efficiently promote viral infection, replication, assembly, and latency, demonstrating their druggable potential by targeting LLPS. In vitro LLPS assays have been widely used to characterize the biophysical properties and study the biochemical functions of phase-separated proteins. By simulating the intracellular environment, these assays can elucidate how cellular or viral proteins form dynamically reversible condensates via LLPS. Here, we describe the procedure of the in vitro LLPS assay for viral proteins, which can also be applied to study the LLPS properties of cellular proteins.