Roots of most terrestrial plants show hydrotropic curvature when exposed to a moisture gradient. Though this root response is difficult to visualize in the soil habitat, there are reports of hydrotropism as an inherent response of primary roots of different plant species, such as Arabidopsis thaliana, Pisum sativum, and Zea mays L., from in vitro system studies. Many plant species use hydrotropism as a mechanism of avoidance to water stress. The actively growing root tip has the ability to change its direction towards greater water availability by differential growth in the elongation zone. The study of this tropic response has been challenged by the interaction of gravitropism, thigmotropism and possibly phototropism. It is hard to visualize hydrotropic curvature in vitro unless all other stimuli are neutralized by the presence of a moisture gradient. In this chapter, we describe methods for preparation of two assay systems used to visualize hydrotropic curvature in the primary roots of Arabidopsis and one moisture gradient system used for maize root seedlings.