Profiling chromatin accessibility at the single-cell level provides critical information about cell type composition and cell-to-cell variation within a complex tissue. Emerging techniques for the interrogation of chromatin accessibility in individual cells allow investigation of the fundamental mechanisms that lead to the variability of different cells. This protocol describes a fast and robust method for single-cell chromatin accessibility profiling based on the assay for transposase-accessible chromatin using sequencing (ATAC-seq). The method combines up-front bulk Tn5 tagging of chromatin with flow cytometry to isolate single nuclei or cells. Reagents required to generate sequencing libraries are added to the same well in the plate where cells are sorted. The protocol described here generates data of high complexity and excellent signal-to-noise ratio and can be combined with index sorting for in-depth characterization of cell types. The whole experimental procedure can be finished within 1 or 2 d with a throughput of hundreds to thousands of nuclei, and the data can be processed by the provided computational pipeline. The execution of the protocol only requires basic techniques and equipment in a molecular biology laboratory with flow cytometry support.