For many years, histones were considered passive structural components of eukaryotic chromatin. Meanwhile it has been proven that histones also participate in gene regulation and repression via post-translational modification. The multitude of these post-translational modifications and the existence of numerous histone variants require particular separation strategies for their analysis, a prerequisite for studying biological processes. The most widely utilized techniques for the separation of histones, namely PAGE, HPCE, RP-HPLC, and hydrophilic Interaction LC, are reviewed here. Problems inherent to the analysis of histones owing to their unique physical and chemical properties along with advantages and shortcomings of particular methods are discussed.