Fluorescence spectroscopy has long been a popular method for protein studies from which researchers have garnered a wealth of biophysical information (1,2). Several specific fluorescence methods have been recently well reviewed (3–5) and readers are encouraged to seek out these references for theory and methods complimentary to those presented in this chapter. The basic selling features for the general use of this tool in biological systems include the relatively low concentrations of sample material required, the occurrence of natural fluorophores in proteins such as tryptophan and tyrosine, the breadth of fluorescence experiments available, and the comparatively simple (and inexpensive) equipment required for most experiments. It is no surprise then that the literature is replete with examples of calcium-binding proteins which have in one way or another been characterized by some fluorescence method. Information available to the researcher includes, but is not limited to, biochemical characteristics such as conformational changes, protein-protein interactions, metal-binding information, membrane localization, long-range distance measurements, and kinetic/dynamic parameters. The majority of this chapter will concentrate on protocols for simple steady-state single-tryptophan fluorescence measurements to probe protein-peptide interactions. References to other fluorescence methods and applications will also be provided.