The　TZM-bl　pseudovirus　assay　is　commonly　used　to　evaluate　the　efficacy　of　neutralizing　antibodies　and　small　molecular　inhibitors　in　HIV-1　research.　Here,　to　determine　the　optimal　measurement　method　for　screening　anti-HIV-1　inhibitors,　we　compared　three　measurement　methods　based　on　firefly　luciferase　and　beta-galactosidase　activities.　The　50%　tissue　culture　infective　doses　(TCID50)　of　the　pseudoviruses　were　determined　using　the　luciferase,　beta-galactosidase　colorimetric,　and　5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside　(X-gal)　staining　assays.　Three　commercial　reverse-transcriptase　inhibitors　(azidothymidine,　nevirapine,　and　lamivudine)　were　tested　as　reference　drugs　to　compare　the　reproducibility,　linear　correlation,　and　half　maximal　inhibitory　concentration　(IC50)　values　determined　using　these　methods.　In　the　TCID50　assay,　the　sensitivity　of　beta-galactosidase　colorimetric　assay　was　almost　562　times　lower　than　that　of　the　other　two　methods.　Reproducible　dose-response　curves　were　obtained　for　the　inhibitors　with　all　methods;　the　IC50　values　of　the　inhibitors　were　not　significantly　different.　Linear　regression　analysis　showed　linear　correlation　between　methods.　Compared　to　the　beta-galactosidase　colorimetric　assay,　the　other　two　methods　have　the　advantage　of　high　sensitivity　and　are　less　affected　by　interference.　In　conclusion,　the　luciferase　and　X-gal　staining　assays,　which　can　be　applied　either　alone　or　combined,　are　recommended　for　anti-HIV-1　inhibitor　screening.　(C)　2016　Elsevier　B.V.　All　rights　reserved.