Information about concentration, molecular structure, binding events, and motion can be obtained using fluorescence spectroscopy methods. Fluorescence polarization spectroscopy is one such method, which studies the relationship between the polarization of light that is used for excitation and light that is subsequently detected from fluorescence. The extent of change of polarization between excitation and emission can be used to study physical processes such as rotational diffusion, extent of denaturation, and orientation at surfaces. In this article Mann and Krull describe the underlying principles to the technique and show how fluorescence polarization spectroscopy has contributed to protein analysis.