Nitric oxide and reactive oxygen species have emerged as important signalling molecules in plants. The half-lives of NO and ROS are very short therefore rapid and precise measurements are required for the understanding biological roles of these redox active species. Various organelles and compartments generate NO and ROS thus it is important to determine precise location of these free radicals in order to understand their signalling roles. Diaminofluorescen (DAF) and fluorescent 2', 7'-dichlorofluorescein (DCF) dyes are employed to determine NO and ROS localisation. The advantage of this approach is that the dyes diffuse precisely to NO and ROS producing sites and generate fluorescence which can be detected by fluorescence- or confocal laser scanning microscopes. However, this technique has its disadvantages; particularly the specificity of the fluorescence signals needs to be established. Therefore, the use scavenger of NO such as cPTIO and ROS such as ascorbate is required to confirm the specificity of the fluorescence signal and ideally, confirmation of data obtained using other methods due to advantage and disadvantage associated with each method (Gupta and Igamberdiev, 2013). Here we describe a method to detect NO and ROS production from Arabidopsis roots in response to infection by Trichoderma, Fusarium using DAF, gas phase Griess reagent assay and DCF fluorescence methods.