Publisher Summary This chapter focuses on the flow cytometric analysis of salmonella-containing vacuoles. Salmonella enterica serovar typhimurium (S. typhimurium) is an enteric pathogen that causes gastroenteritis in humans. In mice, it is the etiologic agent of a systemic infection similar to typhoid fever. Thus it is used as a model to study the pathophysiology of typhoid fever. After uptake, Salmonella resides within a vacuole that successively acquires markers of the early endosomes, the recycling compartment and late endocytic compartments. Salmonella grown under different conditions are needed whether the invasion of phagocytic or non-phagocytic is being used. As Salmonella grown to the log phase induce apoptosis of macrophages, invasion of this cell type requires bacteria grown to stationary phase. Optimized cell invasion is necessary for the analysis of fluorescent vacuoles by flow cytometry. Optimal detection is achieved when vacuoles represent more than 5% of particles. This requires cells infected with between 1 and 10 fluorescent bacteria. Many bacterial pathogens reside within host cells, either transiently or throughout an infection, and this intracellular lifestyle is often a key component to disease.