Mast cells are a critical component of host defense against bacterial infections. Activation of these cells during infection induces both innate and adaptive aspects of protective immunity needed for the elimination of the bacteria and survival of the host. These functional roles for the mast cell have been principally characterized using two in vivo models of acute bacterial infection featuring Gram-negative pathogens such as Escherichia coli . Here, we present basic protocols for the identification of mast cell-dependent biological functions during bacterial infection. These include the use of mast cell-deficient mice, the identification of mast cells in tissue, the culture of uropathogenic E. coli , and the basic analysis of mast cell-dependent functions in the peritoneal cavity and footpad models of bacterial pathogenesis.