Bacterial aerotaxis is a rapid response towards or away from oxygen. Here we report on the use of computer-assisted motion analysis coupled to flash photolysis of caged oxygen to quantify aerotactic responses in bacteria. The caged compound (μ-peroxo)(μ-hydroxo)bis[bis(bipyridyl) cobalt(III)] perchlorate liberates molecular oxygen upon irradiation with near-UV light. A mixture of cells and the caged oxygen compound was placed in a capillary tube and challenged by discrete stimuli of molecular oxygen produced by photolysis. We then recorded the rate of change of direction (rcd) as an estimate of tumble frequency in response to liberated oxygen and measured the signal processing (excitation) times in Bacillus subtilis, Bacillus halodurans and Escherichia coli. This computer-assisted caged oxygen assay gives a unique physiological profile of different aerotaxis transducers in bacteria.