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The T box mechanism is a gene expression controlling mechanism in various Gram-positive bacteria species. Recognition of the T box antiterminator and the tRNA is an essential step for the gene expression, which might function as a potential drug target for the development of antibacterial drugs. Libraries of ligands, includingaminoglycosides, triazoles and oxazolididones, were tested for targeting effects on the T box antiterminator and inhibition effects on the formation of the tRNA-antiterminator complex. Mg2+ and spermidine, as cofactors for the T box antitermination, were also investigated for their effects on the recognition of tRNA and the T box antiterminator model.Fluorescence screening assay, fluorescence anisotropy screening assay and fluorescence binding assay were developed and/or optimized for testing ligand interactions with the T box antiterminator model and also ligand inhibition effects on the formation of the tRNA-antiterminator complex. Four ligands show high potentials for the drug discovery purpose in reference to the combinational results of the three assays: IAB-3, IAB-4, RLB-42 and RLB-38.Fluorescence techniques, including fluorescence binding and fluorescence anisotropy were applied for monitoring the tRNA-T box antiterminator interaction at variable Mg2+/spermidine conditions. Thermodynamic characterization of the Mg2+ and spermidine effects on the tRNA-antiterminator complex formation was performed by the Isothermal Titration Calorimeter (ITC). ITC results indicated that under in vitro condition, different Mg2+ levels can facilitate the formation of the discriminator base pair between the tRNA and the antiterminator model and induce conformational changes in the complex, which might provide thermodynamic explanation for the antitermination initiated by the tRNA-T box antiterminator recognition in the T box mechanism. Application of spermidine might serve as a solution to lower the high Mg2+ requirement in the in vitro research on the T box antitermination mechanism.