Mapping the position of nucleosomes in vivo is key to our understanding of chromatin-based processes such as gene transcription, DNA replication, and repair. Methods based on micrococcal nuclease (MNase) digestion of chromatin are widely used to map nucleosomes, but these methods suffer from some limitations. More recently, nucleosome mapping methods that take advantage of our ability to convert cysteine residues—carefully inserted in histone proteins—into nucleases provide alternatives to MNase-based assays. Here, we provide a detailed protocol for the mapping of nucleosomes via H3Q85C-directed DNA cleavage in Saccharomyces cerevisiae.