Several methods for determination of the secondary structure of proteins by spectroscopic measurements are reviewed. Circular dichroism (CD) spectroscopy provides rapid determinations of protein secondary structure with dilute solutions and a way to rapidly assess conformational changes resulting from addition of ligands. Both CD and Raman spectroscopies are particularly useful for measurements over a range of temperatures. Infrared (IR) and Raman spectroscopy require only small volumes of protein solution. The frequencies of amide bands are analyzed to determine the distribution of secondary structures in proteins. NMR chemical shifts may also be used to determine the positions of secondary structure within the primary sequence of a protein. However, the chemical shifts must first be assigned to particular residues, making the technique considerably slower than the optical methods. These data, together with sophisticated molecular modeling techniques, allow for refinement of protein structural models as well as rapid assessment of conformational changes resulting from ligand binding or macromolecular interactions. A selected number of examples are given to illustrate the power of the techniques in applications of biological interest.