Strong promoters cloned into transcriptional fusion vectors can adversely affect plasmid copy number. In this study, we investigated the use of transcriptional repressors, lacI and tetR, to stabilize the copy number of plasmids containing the lacUV5 and tetA promoters, respectively. Repression of these promoters was found to prevent plasmid copy number variation. Transcriptional strength of these promoters, when cloned into transcriptional fusion vectors, was determined by measuring the rate of synthesis after derepression with inducer. By using this approach, promoter strength can be accurately measured in vivo, without the need to compensate for copy number variation.