Plants are sessile organisms that constantly need to adapt to their changing environment. The root is exposed to numerous environmental signals ranging from nutrients and water to microbial molecular patterns. These signals can trigger distinct responses including the rapid increase or decrease of root growth. Consequently, using root growth as a read out for signal perception allows for deciphering which cues root senses and how they integrate it. To date, studies measuring root growth responses using large numbers of roots are often limited either by the lack of high-throughput image acquisition, scalable analysis methods, and by low spatiotemporal resolution. We developed the Root Walker pipeline, which utilizes automated microscopes to acquire images of many roots exposed to controlled treatments with high-spatiotemporal-resolution in conjunction with a fast and automated image analysis software. We demonstrate the power of this pipeline by quantifying fast root growth rate responses in the order of minutes upon treatments with natural auxin, and by quantifying slower root growth rate responses in the order of hours upon treatment with two mitogen-associated protein kinase cascade inhibitors. We find a concentration dependent root growth response to auxin and expose the specificity of one MAPK inhibitor for affecting root growth. Overall, the Root Walker toolkit is a relevant tool for the plant community to conduct large-scale screening of root growth changes for a variety of purposes including genetic screens for root sensing and response mechanisms.