Publisher Summary This chapter describes the picosecond spectroscopy and its application to biological systems. To determine the timewidth of a single picosecond pulse or pulse train, a two-photon fluorescence technique is used. This method uses the simultaneous absorption of two photons via a virtual transition in a dye selected for its high absorption cross-section at the two-photon energy and a subsequent high quantum yield of fluorescence. The generation of the picosecond pulses is achieved in a cavity formed by 100% and ∼50% reflecting mirrors. The emphasis of the chapter is given to the technological aspects and means for detection, display, and processing of data in the picosecond region. The identification and kinetics of short-lived intermediates in photosynthetic (bacteriochlorophyll) and visual (rhodopsin) processes are used as illustrations of the potential application of picosecond spectroscopy for the elucidation of the primary processes of importance in biology.