Here we provide a step-by-step protocol for the application of synthetic theophylline-dependent riboswitches for conditional gene expression in Streptomyces coelicolor. Application of the method requires a sequence of only ~ 85 nt to be inserted between the transcriptional start site and the start codon of a gene of interest. No auxiliary factors are needed. All tested riboswitch variants worked well in concert with the promoters galP2, ermEp1, and SF14. Moreover, they allowed theophylline-dependent expression not only of the heterologous β-glucuronidase reporter gene but also of dagA, an endogenousagarase gene.