Staphylococcus aureus is a leading cause of human infections worldwide and causes a variety of diseases ranging in severity from mild to life-threatening. The ability of S. aureus to cause disease is based in part on its ability to subvert the innate immune system. Advances in genome-wide analysis of host-pathogen interactions have provided the necessary tools to investigate molecular factors that directly contribute to S. aureus pathogenesis. This chapter describes methods to analyze gene expression in S. aureus during interaction with human neutrophils.