Nutrient uptake by roots of mature trees is difficult to measure accurately under field conditions using existing methods. In this review, we discuss current techniques for measuring uptake at the root surface including excised roots, isotopic tracers, autoradiography, depletion, and lysimeters. Although these methods have provided many insights, each has drawbacks. Estimates of uptake are affected by the sampling scheme, experimental conditions, whether roots are excised or not, concentrations of ions, and the rate of efflux of ions. Microbes and mycorrhizas can also affect estimates of uptake. A greater focus on methods development is critical to advancing our understanding of nutrient uptake of mature trees under conditions representative of those in the field.