Open or accessible chromatin typifies euchromatic regions and helps define cell type-specific transcription programs. DNA replication massively disorders chromatin composition and structure, and how accessible regions are affected by and recover from this disruption has been unclear. Here, we present repli-ATAC-seq, a protocol to profile accessible chromatin genome-wide on replicated DNA starting from 100,000 cells. In this method, replicated DNA is labeled with a short 5-ethynyl-2′-deoxyuridine (EdU) pulse in cultured cells and isolated from a population of tagmented fragments for amplification and next-generation sequencing. Repli-ATAC-seq provides high-resolution information on chromatin dynamics after DNA replication and reveals new insights into the interplay between DNA replication, transcription, and the chromatin landscape.