The authors demonstrate that the fluorescence lifetime of certain fluorescent labels is a useful parameter to detect affinity binding between biotin and streptavidin, as well as between biotinylated bovine serum albumin and streptavidin. The assay is performed in a microplate format, and lifetimes are determined using dye laser-induced fluorescence. Four fluorescent labels are presented that undergo a significant change in their lifetime upon affinity binding. The scheme. referred to as the fluorescence lifetime affinity assay, has several attractive features in that it requires single labeling only, represents a homogeneous assay, allows each of the 2 binding partners to be labeled, and is compatible with the standard microwell formats used in hight-throughput screening.