Scientists have long sought technical approaches that would allow them to study the real-time behavior of cellular functions at the subcellular level. Fluorescence, which is the emission of photons that occur as the electrons in a chromophore decay from an excited state back to the ground state, has provided a tool by which scientists can examine various cellular properties. This chapter discusses how fluorescence can be used as a spectroscopic ruler to measure distances among cellular components and gain information about the interactions of these components on the molecular level. This method, known as “resonance energy transfer (RET),” is founded on the fact that a fluorophore (donor) in an excited state may transfer its excitation energy to a neighboring chromophore (acceptor) nonradiatively through dipole–dipole interactions. This process requires some spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor and, for a given donor-acceptor pair, the efficiency of the transfer process is dependent on their relative orientation and on the distance between them.