The ABACUS1-2 μ (ABscisic Acid Concentration and Uptake Sensor 1-2 μ) and GPS1 (Gibberellin Perception Sensor 1) are direct Förster resonance energy transfer (FRET) biosensors that can be used to measure hormone levels in planta. We provide detailed protocols to image FRET biosensors under exogenously applied hormones in roots, either as a single time point or for treatment time courses before and after hormone application. A new, free, open-source analysis toolset for Fiji is introduced and used to get full 3D segmentation of images of nuclear localized FRET biosensors and calculate emission ratios on a per nucleus basis allowing in-depth analysis of biosensor data.