Fluorescence spectroscopy may serve as a universal tool for the study of protein–ligand interactions. Applications of fluorometry have made use of various aspects of fluorescence such as intensity, emission and excitation spectra, lifetime, quantum yield, polarization state, and anisotropy, as well as energy transfer and other electronic phenomena. An experimentalist has to consider each of these characteristics carefully, frequently in combination with each other, for the analysis of protein–ligand complexes and for the determination of binding constants. Most of the available techniques are of a rather general nature and a wealth of possibilities exists for their utilization. In this chapter we will provide a short survey of selected techniques that can be used for measuring binding constants and probing protein–ligand interactions. Basic principles and phenomena are discussed followed by experimental considerations and examples of binding constant determination. Emphasis is placed on steady-state techniques that employ the use of intrinsic protein fluorescence, labeled ligands, as well as anisotropy and resonance energy transfer.