Synchrotron-based X-ray fluorescence (XRF) is allowing substantial advances in several disciplines of plant science by allowing the in situ examination of elements within plant tissues. Continual improvements in detector speed, sensitivity, and resolution are increasing the diversity of questions that can be addressed using this technique, including the in situ analysis of elements (such as nutrients or toxicants) within fresh and hydrated tissues. Here, we describe the general principles for designing and conducting experiments for the examination of elemental distributions in plant material using micro-XRF.