Aim:To develop a high-throughput real-time assay based on molecular beaconsto monitor the integrase 3’-processing reaction in vitro and apply it to inhibitorscreening.Methods:The recombinant human immunodeficiency virus (HIV)-1integrase (IN) is incubated with a 38 mer oligonucleotide substrate,a sequenceidentical to the U5 end of HIV-1 long terminal repeats (LTR).Based on the fluores-cence 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 anincrease in fluorescence which can be monitored on a spectrofluorometer.Tooptimize this assay,tests were performed to investigate the effects of substrates,enzyme and the metal ion concentrations on the IN activity and optimal param-eters were obtained.Moreover,2 IN inhibitors were employed to test the perfor-mance of this assay in antiviral compound screening.Results:The fluorescentintensity of the reaction mixture varies linearly with time and is proportional to thevelocity of the 3’-processing reaction.Tests were performed and the results showedthat the optimal rate was obtained for a reaction mixture containing 50 mg/L recom-binant HIV-1 IN,400 nmol/L substrate,and 10 mmol/L Mn~(2+).The IN 3’-processingreaction under the optimal conditions showed a more than 18-fold increase in thefluorescence intensity compared to the enzyme-free control.The IC_(50) values ofthe IN inhibitors obtained in our assay were similar to the values obtained from aradiolabeled substrate assay.Conclusion:Our results demonstrated that this is afast,reliable,and sensitive method to monitor HIV IN 3’-processing reaction andthat it can be used for inhibitor screening. Aim: To develop a high-throughput real-time assay based on molecular beaconsto monitor the integrase 3’-processing reaction in vitro and apply it to inhibitorscreening. Methods: The recombinant human immunodeficiency virus (HIV) -1integrase (IN) is incubated with a 38 mer oligonucleotide substrate, a sequenceidentical to the U5 end of HIV-1 long terminal repeats (LTR). Based on the fluores-cence 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 anincrease 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 param-ents were obtained. Moreover, 2 IN inhibitors were employed to test the perfor-mance 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 recombination of HIV- 1 IN, 400 nmol / L substrate, and 10 mmol / L Mn ~ (2 +). The IN 3’-processing reaction under the optimal conditions showed a more than 18-fold increase in the fluorescence intensity than the enzyme- free control. The IC 50 values ​​of the IN inhibitors obtained in our assay were similar to the values ​​obtained from aradiolabeled substrate assay. Confluence: Our results of that this is a fast, reliable, and sensitive method to monitor HIV IN 3’-processing reaction and it it can be used for inhibitor screening.