A protocol of a recently developed assay is presented in which FACS-based flow cytometry is used in combination with autofluorescent reporter constructs to simultaneously monitor and quantify bacterial growth (root colonization levels) and single cell activity (expression of a plant-beneficial trait) on roots of hydroponically grown plants. The experimental approach is illustrated with an example of a reporter of antifungal gene expression in a plant-beneficial strain of Pseudomonas fluorescens. It can be adapted to the study of other bacteria-root interactions.