Cellular processes involve a multitude of chemical reactions that must be kept in delicate equilibrium to maintain cellular homeostasis. Powerful biophysical techniques are needed to measure the localization and concentration of target molecules as well as to quantify complex molecular processes in model and in vivo systems. Two-photon microscopy and fluorescence correlation spectroscopy (FCS) can measure association and dynamics of appropriate molecules under equilibrium conditions. FCS provides information on motility (diffusion coefficients), concentration (number of particles), association (molecular brightness), and localization (image) of the target molecules. All of this information, in conjunction with computational modeling techniques, can help us to better understand the network of complex molecular interactions, which are at the basis of cellular processes. Fluorescence imaging techniques add the beauty of visualization to the scientific information. Photons emitted by a fluorescent dye are digitized, and the associated spatial information and intensity can be translated into different colors and shades providing the researcher not only with quantitative intensity information but also with spatial resolution and visual comprehension of two- or three-dimensional images. In this Account, we review the use of two-photon excitation microscopy and FCS in the study of lipid-protein interactions. We discuss these new methodologies and techniques, and we present examples of different complexity from qualitative to quantitative, from simple model systems to studies in living cells.