Histone post-translational modifications (PTMs) are key regulators of chromatin architecture and gene expression. Although DIA-based platforms for histone PTM quantification are available, multiplexed targeted assays remain underdeveloped. In addition, bottom-up workflows frequently rely on chemical derivatization, which can introduce variability, increase procedural complexity, and obscure biologically relevant PTMs. Here, we present a derivatization-free parallel reaction monitoring (PRM) workflow for robust, quantitative, and site-specific analysis of histone PTMs. We employed highly efficient ArgC digestion to generate peptides of optimal length for LC–MS/MS without compromising endogenous PTMs, and we optimized chromatographic conditions to achieve isobaric separation and stable retention times. Co-eluting isobaric PTM species were confidently distinguished using site-specific fragment ions. The resulting PRM method enabled sensitive and reproducible detection of histone PTM isoforms across diverse biological systems. To illustrate its utility, we profiled PTM changes in cells expressing K-to-M mutations and in cells treated with the HDAC inhibitor entinostat, yielding results that correlated strongly with antibody-based assays. This PRM platform provides a practical and versatile framework for targeted analysis of histone PTMs identified across epigenetic studies.