Genomic insulators are DNA elements that prevent transcriptional activation of a promoter by an enhancer when interposed. We present a protocol for insulator-seq that enables high-throughput screening of genomic insulators using a plasmid-based massively parallel reporter assay in Drosophila cultured cells. We describe steps for insulator reporter plasmid library generation, transient transfection into cultured cells, and sequencing library preparation and provide a pipeline for data analysis. For complete details on the use and execution of this protocol, please refer to Tonelli et al. ¹ .