The regulation of DNA accessibility by histone modification has emerged as a paradigm of developmental and environmental programming. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a versatile tool to investigate in vivo protein-DNA interaction and has enabled advances in mechanistic understanding of physiologies. The technique has been successfully demonstrated in several plant species and tissues; however, it has remained challenging in woody tissues, in particular complex structures such as perennating buds. Here we developed a ChIP method specifically for mature dormant buds of grapevine (Vitis vinifera cv. Cabernet Sauvignon). Each step of the protocol was systematically optimized, including crosslinking, chromatin extraction, sonication, and antibody validation. Analysis of histone H3-enriched DNA was performed to evaluate the success of the protocol and identify occupancy of histone H3 along grapevine bud chromatin. To our best knowledge, this is the first ChIP experiment protocol optimized for the grapevine bud system.