Fluorescence correlation spectroscopy (FCS) is a minimally invasive real-time fluorescence technique capable of detecting single molecules in vitro and in situ. By recording and correlating the fluctuations in fluorescence intensity measurements, it is possible to obtain information on molecular mobility and diffusion, hydrodynamic radii, local concentrations and photochemical and photophysical properties. By using dual-color cross-correlation spectroscopy, it is possible to monitor highly specific molecular-level interactions such as binding processes and chemical reactions. Recent advances in alternative detection schemes have allowed the extension of these techniques to the monitoring of slower timescales (e.g. Raster Image Correlation Spectroscopy. - RICS) or higher concentrations (e.g. Total Internal Reflection. - TIR-FCS). Given the versatility of these techniques, they have become commonplace tools used to specifically unravel the spatio-temporal dynamics of macromolecular entities in living biological systems.