Current methods for R-loop mapping need to perform DNA:RNA immunoprecipitation for each sample individually, with consequent limitations in throughput. Here, we develop and validate mDRIP-seq, a multi-sample barcoding and pooling method for R-loop mapping. We show mDRIP-seq performs equivalently as conventional methods, but with the merits of high throughput and cost-efficiency. We also show the simplicity of mDRIP-seq for relative and absolute quantitation of genomic R-loop fractions for multiple samples. Together, mDRIP-seq is a high-throughput and cost-efficient method for R-loop mapping and quantitative assessment and can be widely applied to large-scale dynamic profiles of these important structures for diverse organisms. Overall design: mDRIP-seq development, and R-loop mapping in multiple samples