Reporter-gene activation studies using transient transformation of protoplasts are a powerful tool for the investigation of transcriptional regulation in plants. Here, we perform a comparative analysis of reporter-gene activation sensitivity using an integrated versus a co-transfected reporter-gene construct in Arabidopsis seedling mesophyll protoplasts. The DR5 synthetic auxin-responsive promoter was used to assay the response to auxin treatment and over-expression of activator Auxin Response Factors. We show that sensitivity, as measured by the fold-change in fluorescent-protein reporter-gene expression, is significantly increased by using a co-transfected reporter-gene construct.