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Despite its extensive use as a nitrogen fertilizer, the role of urea as a directly accessible nitrogen source for crop plants is still poorly understood. So far, the physiological and molecular aspects of urea acquisition have been investigated only in a few plant species highlighting the importance of a urea transporter in roots, DUR3 (Kojima et al., 2007; Wang et al., 2012; Zanin et al., 2014a). Regarding maize plants, a crop that needs a large amount of urea fertilizer, the capability to take up urea via an inducible and high-affinity transport system has been recently characterized (Zanin et al., 2014a; Zanin et al., 2014b). Here, we described a small-scale protocol suitable for the measurement of urea net high-affinity uptake in roots of intact maize plants.