Reactive oxygen species (ROS) are byproducts of fundamental metabolic processes in plants. Abiotic stress, such as drought, elevates production of ROS. Among other ROS in plants, hydrogen peroxide (H2O2) has the longest half-life and is critical for downstream signaling in processes leading to defense against abiotic and biotic stress. Despite the importance of ROS, methods for their absolute quantification in plants are not available. Here we describe the development of a fluorimetric technique using the dye Amplex Red to quantify H2O2 and other ROS in plant tissue. The technique was tested on shoot tissue that was either inoculated with Diplodia pinea (a fungal pathogen that causes tip blight and canker) or left uninoculated, derived from Austrian pine (Pinus nigra) saplings that were either droughted or not. As expected, in the absence of infection we observed significantly higher levels of ROS in the shoots of droughted than non-droughted trees (9.01 ± 1.22 nmol g-1 FW and 4.74 ± 0.77 nmol g-1 FW, respectively). D. pinea infection resulted in significantly lower ROS concentrations than in corresponding mock-inoculated shoots on droughted trees (3.96 ± 0.20 nmol g-1 FW in inoculated vs. 7.90 ± 0.87 nmol g-1 FW in non-inoculated pines), but there were no significant differences in ROS concentrations between inoculated (5.12 ± 0.78 nmol g-1 FW) and non-inoculated (5.89 ± 0.28 nmol g-1 FW) shoots on non-droughted trees. Furthermore, D. pinea inoculation resulted in significantly longer lesions in droughted than non-droughted pines (14.01 ± 0.95 cm and 6.90 ± 0.60 cm, respectively). Our results demonstrate the usefulness of our method in studies investigating complex interactions between abiotic and biotic stress on ROS metabolism in plants.