Chromatin accessibility is directly linked with transcription in eukaryotes. Accessible regions associated with regulatory proteins are highly sensitive to DNase I digestion and are termed DNase I hypersensitive sites (DHSs). DHSs can be identified by DNase I digestion, followed by high-throughput DNA sequencing (DNase-seq). The single-base-pair resolution digestion patterns from DNase-seq allows identifying transcription factor (TF) footprints of local DNA protection that predict TF-DNA binding. The identification of differential footprinting between two conditions allows mapping relevant TF regulatory interactions. Here, we provide step-by-step instructions to build gene regulatory networks from DNase-seq data. Our pipeline includes steps for DHSs calling, identification of differential TF footprints between treatment and control conditions, and construction of gene regulatory networks. Even though the data we used in this example was obtained from Arabidopsis thaliana, the workflow developed in this guide can be adapted to work with DNase-seq data from any organism with a sequenced genome.