Chromatin is a macromolecular complex composed of DNA and histones that regulate gene expression and nuclear architecture. The concerted action of DNA methylation, histone post-translational modifications and chromatin-associated proteins control the epigenetic regulation of the genome, ultimately determining cell fate and the transcriptional outputs of differentiated cells. Deregulation of this complex machinery leads to disease states, and exploiting epigenetic drugs is becoming increasingly attractive for therapeutic intervention. Mass spectrometry (MS)-based proteomics emerged as a powerful tool complementary to genomic approaches for epigenetic research, allowing the unbiased and comprehensive analysis of histone post-translational modifications and the characterization of chromatin constituents and chromatin-associated proteins. Furthermore, MS holds great promise for epigenetic biomarker discovery and represents a useful tool for deconvolution of epigenetic drug targets. Here, we will provide an overview of the applications of MS-based proteomics in various areas of chromatin biology.