cis-Regulatory analysis (CRA) is the precise identification of the cis-acting genomic sequences regulating gene transcription. As such, CRA provides essential mechanistic insight into key biological processes such as development. The first phase of this protocol involves identification of a large (approximately 100 kb) clone of genomic material surrounding the gene of interest and use of this clone to establish a reliable and unambiguous reporter assay. In the second phase, phylogenetic footprinting is used to identify candidate regulatory modules; these genomic sequences are then recursively tested for reporter activity. In the final phase, potential transcription factor binding sites are identified and disrupted in reporter constructs for individual testing. The strengths of this method reflect the use of large clones containing all relevant genomic regulatory sequences to establish a reporter assay with high fidelity. Given these foundational elements, predicted or suspected regulatory inputs can be rigorously tested and novel regulatory inputs identified. Although the expected time line varies greatly with the depth of information required, results may be obtained in as little as 4-6 months, but more detailed analyses will require several years to complete.