Aim:　To　develop　a　novel　high-throughput　format　assay　to　monitor　the　integrase　(IN)　strand　transfer　(ST)　reaction　in　vitro　and　apply　it　to　a　reaction　character　study　and　the　identification　of　antiviral　drugs.　Methods:　The　donor　DNA　duplex,　with　a　sequence　identical　to　the　U5　end　of　HIV-1　long　terminal　repeats,　is　labeled　at　its　5＇　end　with　biotin　(BIO).　The　target　DNA　duplex　is　labeled　at　its　3＇　end　with　digoxin　(DIG).　IN　mediates　the　integration　of　donor　DNA　into　target　DNA　and　results　in　a　5＇　BIO　and　3＇　DIG-labeled　duplex　DNA　product.　Streptavidin-coated　magnetic　beads　were　used　to　capture　the　product,　and　the　amount　of　DIG　was　measured　as　the　ST　reaction　product.　The　assay　was　optimized　in　96-well　microplate　format　for　high-throughput　screening　purpose.　Moreover,　the　assay　was　applied　in　a　ST　reaction　character　study,　and　the　efficiency　of　the　assay　in　the　identification　of　antiviral　compounds　was　tested.　Results:　The　end-point　values,　measured　as　absorbance　at　405　nm　was　approximately　1.5　for　the　IN-mediated　ST　reaction　as　compared　with　no　more　than　0.05　of　background　readings.　The　ST　reaction　character　and　the　half　maximal　inhibitory　concentration　(IC50)　values　of　2　known　IN　inhibitors　obtained　in　our　assay　were　similar　to　previously　reported　results　using　other　assays.　The　evaluation　parameter　Z＇　factor　for　this　assay　ranged　from　0.6　to　0.9.　Conclusion:　The　assay　presented　here　has　been　proven　to　be　rapid,　sensitive,　and　specific　for　the　detection　of　IN　ST　activity,　the　reaction　character　study,　as　well　as　for　the　identification　of　antiviral　drugs　targeting　IN.