This chapter provides a comprehensive description of this streak camera-based fluorescence lifetime imaging microscopy (FLIM) imaging (StreakFLIM) system and contrasts its performance with that of the other currently available time-domain FLIM systems. The FLIM methods are relatively insensitive to artifacts that can affect spectroscopic characteristics of fluorophores and can provide distinct lifetimes characteristic of the individual fluorophores attached to each protein (for example, donor and acceptor). The main component of the StreakFLIM system is the streak camera, also known as streakscope, a device that can measure ultrafast light phenomena with a temporal resolution as low as 2 picoseconds (ps). In addition to intensity and time information, the streakscope can be used to obtain either spatial or wavelength information. The streak camera has good radiant sensitivity over a wide bandwidth of wavelengths, making it suitable for both single-photon and multiphoton imaging applications without compromising detection sensitivity. While in the conventional time-domain FLIM, multigate detection with a gated microchannel plate image intensifier and a charge-coupled device (CCD) imaging camera is used.