Aptamers are single-stranded nucleic acid molecules forming well-defined 3D structures. Aptamers typically bind to their ligands with high affinity and specificity. They are capable of interacting with various kinds of ligands: ions, small molecules, peptides, proteins, viruses, bacteria, and even cells. Therefore, aptamers are in widespread use as sensor molecules or as targeting agents in diagnostics and pharmaceutics. As a prerequisite for their use in these economic high-value areas, aptamers must be studied in detail with respect to different biophysical characteristics. Of central importance are basic binding parameters of the aptamer-target interaction, such as binding affinity and kinetics. Numerous biophysical methods with different features, characteristics, and capabilities are used in the field today for this purpose.This chapter provides an overview of the current state-of-the-art technologies for studying interactions between aptamers and targets and discusses their advantages as well as drawbacks. Furthermore, essential aspects influencing any aptamer characterization strategy will be presented. Finally, issues of comparability of binding data between different aptamer characterization technologies will be discussed. Graphical Abstract.