A new generation of reagents that report on specific molecular events in living cells, called fluorescent protein biosensors, has evolved from in vitro fluorescence spectroscopy and fluorescent analogue cytochemistry. Creative designs of fluorescent protein biosensors to measure the molecular dynamics of macromolecules, metabolites, and ions in single cells emerge from the integrative use of contemporary synthetic organic chemistry, biochemistry, and molecular biology. Future advances in fluorescent probe design, computer-driven optical instrumentation, and software will allow us to engineer endogenous cellular components that localize and function as reporters of their activities, thus moving molecular measurement beyond the single cell to living tissues and the whole organism.