Legionella pneumophila infects and replicates in environmental protozoa and metazoan macrophages within a specific vacuole. The infection of phagocytes by L. pneumophila can be assessed by an agar plating assay or by fluorescence microscopy. Here, we describe the analysis of Legionella infection by automated flow cytometry using wild-type and mutant bacteria that constitutively produce the green fluorescent protein (GFP). Advantages of the flow cytometry technique include (1) a software-assisted multiple parameter analysis of Legionella infections in real-time at distinct stages of the infection cycle, (2) the simultaneous and fast acquisition of a high number of data points, and (3) a characterization of the infecting bacteria in parallel with the infected host cells.