In the last few years fluorescence microscopy has become a very useful technique to study the lateral structure of membranes. Several experimental strategies using fluorescence microscopy techniques (epifluorescence, confocal and two-photon excitation) have appeared in the literature employing GUVs as model systems. The advantages and disadvantages of the different experimental approaches, including the use of different fluorescent probes, were discussed in this chapter. In particular, the advantages in using the polarity-sensitive probe LAURDAN as a fluorescent marker under a two-photon-excitation fluorescence microscope to study not only artificial lipid mixtures but also natural lipid extracts and cell membranes was addressed. The visual information obtained with these techniques is extremely useful to correlate the lateral structure of compositionally simple membranes with that observed in compositionally complex mixtures such as biological membranes.