In this article, we introduce the real-time cellular force microscope (RT-CFM), a high-throughput microrobotic platform for mechanical stimulation and characterization of single cells. We developed computer vision algorithms that fully automate the positioning of target cells and localization of the sensor tip. The control and acquisition architecture dramatically increases the accuracy, speed, and reliability of force measurements. Pollen tubes provide an ideal model system for the study of plant mechanics at the single-cell level. To quantitatively obtain the physical properties of the plant cell wall, we generated topography and stiffness measurements from 3D scans of living, growing pollen tubes. We report techniques for real-time monitoring and analysis of intracellular calcium fluxes during mechanical intervention. Our platform is compatible with various imaging systems and enables a powerful screening technology to facilitate biomechanical and morphological characterization of developing cells.