There is an urgent need for new drugs to combat pathogenic viruses. Most existing antivirals inhibit virus attachment/entry or target key enzymes, and new antiviral targets are needed to develop novel treatments and counter antiviral resistance. A key process in the replication cycle of many viruses is the formation of dynamic organelles called viral factories. Multiple lines of evidence suggest that viral factories may form via liquid-liquid phase separation (LLPS), including those that support replication of SARS-CoV-2, influenza, measles, and respiratory syncytial virus. Targeting LLPS is thus an emerging paradigm that may underlie the discovery of novel, broad-spectrum antivirals. This project will focus on dissecting the physicochemical properties of viral RNA-binding proteins that form complex biomolecular condensates to understand how they nucleate viral factories and support viral replication. This will lead to the identification of new therapeutic targets. Central to this proposal will be mass spectrometry (MS)-based proteomics methods combined with machine learning approaches and advanced microscopy and microfluidics techniques that will be used to bridge the gap between in vitro and in cellulo studies of viral factory formation. The insights gained from the work will underlie our search for compounds-modulators of LLPS that could be used for future antiviral therapies.