Cells are the complex product of gene expression programs that involve the coordinated transcription of thousands of genes controlled by -regulatory elements (-REs). Therefore, identification of -REs is the key to decipher the mechanisms underlying the regulation of gene expression. Here, we describe a simple and time-effective protocol of fine-mapping -REs by using an electrophoresis mobility shift assay (EMSA)-based, , high-throughput (HTP) technique called regulatory element-sequencing (Reel-seq). Reel-seq can be applied to identify -REs at a high resolution and sensitivity over large genome regions, in a systematic and continuous manner. It can be used to prioritize candidate -REs as a complement to the existing approaches, such as massive parallel reporter assay (MPRA), chromatin immunoprecipitation DNA-sequencing (ChIP-seq), and the assay for transposase-accessible chromatin sequencing (ATAC-seq). NE: nuclear extract; NGS: next generation sequencing.