Bacteria, in common with other organisms that take advantage of aerobic respiration, generate and accumulate reactive oxygen species (ROS) that damage DNA, fatty acids, and proteins. In addition, intracellular pathogens like Salmonella enterica are exposed to an oxidate burst produced by host macrophages. The relative ability of aerobically growing bacteria to withstand oxidative stress and eliminate ROS has a large impact of their fitness in vitro and in vivo. Methods are described here to measure the viability and relative fitness of bacteria in the presence of hydrogen peroxide. A protocol for the determination of catalase activity, an important part of the ROS detoxification process, is also described.