Plant roots sense salt gradients in soil to avoid saline environments through halotropism. Here, we present a protocol to study halotropism with an optimized split-agar system that simulates the salt gradient in soil. We describe steps for preparation of the split-agar system, measurement of Na⁺, and observation of root bending. We then detail segmentation of root cells and visualization of microtubules and cellulose synthases. This system is simple to operate and has broader applications, such as hydrotropism and chemotropism. For complete details on the use and execution of this protocol, please refer to Yu et al. (2022).¹