Stomata embedded in the epidermis of terrestrial plants are important for CO₂ absorption and water transpiration, and are possible points of entry for pathogens. Thus, the regulation of stomatal apertures is extremely important for the survival of plants. Furthermore, stomata can respond via accurate change of stomatal apertures to a series of extracellular stimuli such as phytohormones, pathogens, ozone, drought, humidity, darkness, CO₂, visible light and UV-B radiation, so stomatal bioassay is widely used to dissect signal transduction mechanisms of plant cells in responses to multiple stimuli. This protocol describes how to measure stomatal apertures in leaves of model plant Arabidopsis thaliana under multiple treatments.