Aim:　To　develop　a　high-throughput　real-time　assay　based　on　molecular　beacons　to　monitor　the　integrase　3＇-processing　reaction　in　vitro　and　apply　it　to　inhibitor　screening.　Methods:　The　recombinant　human　immunodeficiency　virus　(HIV)-1　integrase　(IN)　is　incubated　with　a　38　mer　oligonucleotide　substrate,　a　sequence　identical　to　the　U5　end　of　HIV-1　long　terminal　repeats　(LTR).　Based　on　the　fluorescence　properties　of　molecular　beacons,　the　substrate　is　designed　to　form　a　stem-loop　structure　labeled　with　a　fluorophore　at　the　5＇　end　and　a　quencher　at　the　3＇　end.　IN　cleaves　the　terminal　3＇-dinucleotide　containing　the　quencher,　resulting　in　an　increase　in　fluorescence　which　can　be　monitored　on　a　spectrofluorometer.　To　optimize　this　assay,　tests　were　performed　to　investigate　the　effects　of　substrates,　enzyme　and　the　metal　ion　concentrations　on　the　IN　activity　and　optimal　parameters　were　obtained.　Moreover,　2　IN　inhibitors　were　employed　to　test　the　performance　of　this　assay　in　antiviral　compound　screening.　Results:　The　fluorescent　intensity　of　the　reaction　mixture　varies　linearly　with　time　and　is　proportional　to　the　velocity　of　the　3＇-processing　reaction.　Tests　were　performed　and　the　results　showed　that　the　optimal　rate　was　obtained　for　a　reaction　mixture　containing　50　mg/L　recombinant　HIV-1　IN,　400　nmol/L　substrate,　and　10　mmol/L　Mn2+.　The　IN　3＇-processing　reaction　under　the　optimal　conditions　showed　a　more　than　18-fold　increase　in　the　fluorescence　intensity　compared　to　the　enzyme-free　control.　The　IC50　values　of　the　IN　inhibitors　obtained　in　our　assay　were　similar　to　the　values　obtained　from　a　radiolabeled　substrate　assay.　Conclusion:　Our　results　demonstrated　that　this　is　a　fast,　reliable,　and　sensitive　method　to　monitor　HIV　IN　3＇-processing　reaction　and　that　it　can　be　used　for　inhibitor　screening.