By providing simultaneously structural and dynamic information, fluorescence spectroscopy is an extremely useful tool for the study of proteins and peptides. The structural information obtained by fluorescence spectroscopy methodologies is especially important in the study of systems for which crystallography data is not available, as it is the case of the large majority of membrane proteins. Furthermore, the dynamic information is hardly accessible by crystallography studies. Thus, for most of these applications, fluorescence spectroscopy is the only technique that can probe the time scale (time-resolved fluorescence, fluorescence anisotropy) or the topological range (energy transfer, fluorescence quenching) of the molecular events. The exclusive use of peptide and protein intrinsic fluorescence (tryptophan, tyrosine and phenylalanine residues) combines the advantages of the non-invasive techniques, with a good sensitivity (essential for most of the membrane protein studies). Examples of the different fluorescence spectroscopy methodologies used on membrane proteins and peptides studies will be addressed; Namely, spectral characterization, fluorescence lifetime, fluorescence anisotropy, energy transfer (homo- and heterotransfer), fluorescence quenching and red-edge excitation shift.