Background: Reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anions significantly accumulate during biotic and abiotic stress and cause oxidative damage and eventually cell death. There is accumulating evidence that ROS are also involved in regulating beneficial plant-microbe interactions, signal transduction and plant growth and development. Due to the relevance of ROS throughout the life cycle and for interaction with the multifactorial environment, the physiological phenotyping of the mechanisms controlling ROS homeostasis is of general importance.Results: In this study, we have developed a robust and resource-efficient experimental platform that allows the determination of the activities of the nine key ROS scavenging enzymes from a single extraction that integrates posttranscriptional and posttranslational regulations. The assays were optimized and adapted for a semi-high throughput 96-well assay format. In a case study, we have analyzed tobacco leaves challenged by pathogen infection, drought and salt stress. The three stress factors resulted in distinct activity signatures with differential temporal dynamics. Conclusions:This experimental platform proved to be suitable to determine the antioxidant enzyme activity signature in different tissues of monocotyledonous and dicotyledonous model and crop plants. The universal enzymatic extraction procedure combined with the 96-well assay format demonstrated to be a simple, fast and semi-high throughput experimental platform for the precise and robust fingerprinting of nine key antioxidant enzymatic activities in plants.