Plants have been reported to contain a large set of aquaporins (38 for Arabidopsis), which has been divided into four subfamilies on the basis of similarities in their amino acid sequences. They belong to the large superfamily of major intrinsic proteins (MIP), which was the basis for the nomenclature PIP, TIP, and NIP, also indicating the subcellular localization plasma membrane, tonoplast, and nodule of the respective founding member. The fourth subfamily of small and basic intrinsic proteins is not well characterized so far. The increasing number of reports dealing with various aspects of plant aquaporins is starting to advance our understanding of aquaporin biology in plants. Fundamental questions include: what is the basic function of the different plant aquaporins, what is their primary substrate, and what is the consequence of function/malfunction of a particular aquaporin for the overall function of the plant? Biochemical and biophysical techniques can be employed to get information on the basic functional characteristics of plant aquaporins. An impressive set of techniques has been used to study aquaporin function on molecular, subcellular, and cellular levels in plants, as well as in heterologous expression systems. The physiological role of aquaporins in plants is much less well understood, but reports unraveling the physiological role of aquaporins, mainly employing genetic techniques and functional measurement on the whole plant level, are emerging. The goal of this chapter is to give an overview on the applied methods, together with some exemplary findings.