To investigate the impact of a host factor on viral replication, conventional techniques involve analyzing viral replication within a homogeneous cell population engineered to uniformly express the host factor. In this chapter, we present a method to explore the intricate dynamics between host factors and vaccinia virus (VACV) infection using flow cytometry, which enables the rapid, multi-parametric analysis of individual cells in solution. Cultured cells are transfected to express a host factor fused with a fluorescent protein, followed by infection with a VACV encoding a different fluorescent reporter. Subsequently, cells are washed, fixed, and filtered for flow cytometry analysis. The data are then analyzed with flow cytometry analysis software to evaluate the influence of the host factor on various infection parameters, including the stage of viral infection and viral replication level. This method allows for the precise investigation of proviral or antiviral factor expression in relation to viral replication within a heterogeneous population of cells.