The orchestrated regulation of hundreds of genes responding in a temporal, spatial, and conditional expression is in part mediated by the transient binding of transcription factors to their specific DNA motifs. The analysis of these cis-regulatory DNA sequences is still a challenging task. Therefore, the combination of the transient protoplast expression assay with computer aided sequence analysis is a preferred method for regulatory sequence analysis. The protocols given here describe the use of a reporter gene plasmid to investigate the effects of a transiently co-expressed transcription factor gene in planta. As a number of bioinformatic analysis tools for cis-elements are publicly available, we suggest a workflow for the electronic analysis of promoter sequences. This is in particular difficult, as most analysis programs have not been developed for the investigation of single sequences. We provide insight into bioinformatics tools that investigate cis-element presence, their distribution, and implications for further functional analyses.