We identified the first Spt5-Pol II inhibitors (SPIs). SPIs faithfully reproduced Spt5 knockdown effects on proximal-promoter-pausing, NF-κB activation and the expanded-repeat huntingtin gene in neuronal cells. Using SPIs we identified Spt5 target genes that responded with profoundly diverse kinetics and a novel regulatory element of proximal-promoter-pausing. To obtain a genome-wide view of the impact of SPIs on transcription, we performed Global run-on sequencing (GRO-seq) using nuclei of HeLa cells treated with either DMSO (control) or SPI-21. Overall design: Nuclei were isolated from HeLa cells pre-treated with either DMSO (control) or SPI-21 (50μM) for 1.5 hours