homogeneous, mix-and-read tr-fret assays offer advantages over other biomolecular screening assays, such as fluorescence polarization (fp) or trf assays. in fp assays, background fluorescence due to library compounds is normally depolarized and background signal due to scattered light (e.g. precipitated compounds) is normally polarized. depending on the assay configuration, either case can lead to a false positive or false negative result. however, because the donor species used in a tr-fret assay has a fluorescent lifetime that is many orders of magnitude longer than background fluorescence or scattered light, emission signal resulting from energy transfer can be measured after any interfering signal has completely decayed. tr-fret assays can also be formatted to use limiting receptor and excess tracer concentrations (unlike fp assays), which can provide further cost savings. in the case of trf assays, a wash step is required to remove unbound fluorescent reagents prior to measuring the activity signal of the assay. this increases reagent use, time to complete the assay, and limits the ability to miniaturize the system (e.g. converting from a 384-well microtiter plate to a 1536-well plate). tr-fret assays take advantage of the required proximity of the donor and acceptor species for generation of signal.