Epigenetic regulation of gene expression is, at least in part, mediated by histone modifications. PTMs of histones change chromatin structure and regulate gene transcription, DNA damage repair, and DNA replication. Thus, studying histone variants and their modifications not only elucidates their functional mechanisms in chromatin regulation, but also provides insights into phenotypes and diseases. A challenge in this field is to determine the best approach(es) to identify histone variants and their PTMs using a robust high-throughput analysis. The large number of histone variants and the enormous diversity that can be generated through combinatorial modifications, also known as histone code, makes identification of histone PTMs a laborious task. MS has been proven to be a powerful tool in this regard. Here, we focus on bottom-up, middle-down, and top-down MS approaches, including CID and electron-capture dissociation/electron-transfer dissociation based techniques for characterization of histones and their PTMs. In addition, we discuss advances in chromatographic separation that take advantage of the chemical properties of the specific histone modifications. This review is also unique in its discussion of current bioinformatic strategies for comprehensive histone code analysis.