Very few clinically available antibiotics target bacterial RNA polymerase (RNAP) suggesting it is an underutilized target. The advent of detailed structural information of RNAP holoenzyme (HE) has allowed the design and in silico screening of novel transcription inhibitors. Here, we describe our approach for the design and testing of small molecule transcription inhibitors that work by preventing the interaction between the essential transcription initiation factor σ and RNAP. With the appropriate structural information this approach can be easily modified to other essential protein-protein interactions.