The T box riboswitch is an intriguing potential target for antibacterial drug discovery. Found primarily in Gram-positive bacteria, the riboswitch regulates gene expression by selectively responding to uncharged tRNA to control transcription readthrough. Polyamines and molecular crowding are known to specifically affect RNA function, but their effect on T box riboswitch efficacy and tRNA affinity have not been fully characterized. A fluorescence-monitored in vitro transcription assay was developed to readily quantify these molecular interactions and to provide a moderate-throughput functional assay for a comprehensive drug discovery screening cascade. The polyamine spermidine specifically enhanced T box riboswitch readthrough efficacy with an EC50 = 0.58 mM independent of tRNA binding. Molecular crowding, simulated by the addition of polyethylene glycol, had no effect on tRNA affinity for the riboswitch, but did reduce the efficacy of tRNA-induced readthrough. These results indicate that the T box riboswitch tRNA affinity and readthrough efficacy are intricately modulated by environmental factors.