Bone　marrow　stromal　cell　antigen　2　(BST-2),　also　known　as　CD317　or　tetherin,　has　been　identified　as　a　host　restriction　factor　that　suppresses　the　release　of　enveloped　viruses　from　host　cells　by　physically　tethering　viral　particles　to　the　cell　surface;　however,　this　host　defense　can　be　subverted　by　multiple　viruses.　For　example,　human　immunodeficiency　virus　(HIV)-1　encodes　a　specific　accessory　protein,　viral　protein　U　(Vpu),　to　counteract　BST-2　by　binding　to　it　and　directing　its　lysosomal　degradation.　Thus,　blocking　the　interaction　between　Vpu　and　BST-2　will　provide　a　promising　strategy　for　anti-HIV　therapy.　Here,　we　report　a　NanoLuc　Binary　Technology　(NanoBiT)-based　high-throughput　screening　assay　to　detect　inhibitors　that　disrupt　the　Vpu-BST-2　interaction.　Out　of　more　than　1000　compounds　screened,　four　inhibitors　were　identified　with　strong　activity　at　nontoxic　concentrations.　In　subsequent　cell-based　BST-2　degradation　assays,　inhibitor　Y-39983　HCl　restored　the　cell-surface　and　total　cellular　level　of　BST-2　in　the　presence　of　Vpu.　Furthermore,　the　Vpu-mediated　enhancement　of　pesudotyped　viral　particle　production　was　inhibited　by　Y-39983　HCl.　Our　findings　indicate　that　our　newly　developed　assay　can　be　used　for　the　discovery　of　potential　antiviral　molecules　with　novel　mechanisms　of　action.