An account is given of the recent efforts involving the use of fluorescence spectroscopy to elucidate subtle features of entrapped proteins, including structure, conformational flexibility, accessibility to analyte, stability, and function. The particular areas where spectroscopic methods benefit the study of entrapped proteins are highlighted, and the overall utility of fluorescence spectroscopy for studies of entrapped proteins is discussed. The use of fluorescence to examine factors important to sensor development is also detailed.