Recent advances in laser technology, computational power, and imaging algorithms have extended the application of fluorescence lifetime method into medical imaging. Initial applications of fluorescence lifetime in cell studies focused on autofluorescence lifetimes of endogenous fluorophores. The lifetime signatures of these biomolecules can be altered by changes in normal cell physiology or pathologic conditions. Scientists have accelerated the development of new fluorophores and luminescent materials for lifetime studies. Depending on the ultimate goal of the study, molecular probes with fixed or variable lifetimes can be used. The availability of a commercial small animal imaging system has extended the application of fluorescence lifetime studies in small animal beyond the confines of instrumentation experts. The emergence of newer diffuse optical tomography (DOT)-based lifetime devices will further enhance the quantitative accuracy and imaging depth by this technique.