HIV-1 integrase (HIV-1 IN), a well-validated antiviral drug target, catalyzes multistep reactions to incorporate viral DNA into the genome of the host cell; these include both a 3'-processing (3'P) reaction and a strand transfer reaction. These enzymatic activities can be measured in vitro with short DNA oligonucleotides that mimic a single viral LTR DNA end and purified IN. A highly sensitive and reproducible time-resolved fluorescence (TRF)-based assay for HIV-1 IN 3'P activity is now reported. This assay was optimized with respect to time and concentrations of metal ions, substrate and enzyme. The assay has now been used successfully to measure HIV-1 IN 3'P activity and has been shown to detect the anti-IN activity of several known 3'P inhibition compounds accurately. This assay, which is amenable to high-throughput screening, will be useful for identification of additional HIV-1 IN 3'P inhibitors.