Biological research has always tremendously benefited from the development of key methodology. In fact, itwas the advent of microscopy that shaped our understanding of cells as the fundamental units of life. Microscopic techniques are still central to the elucidation of biological units and processes, but equally important are methods that allow access to the dimension of time, to investigate the dynamics of molecular functions and interactions. Here, fluorescence spectroscopy with its sensitivity to access the single-molecule level, and its large temporal resolution, has been opening up fully new perspectives for cell biology. Herewe summarize the key fluorescent techniques used to study cellular dynamics, with the focus on lipid and membrane systems.