Histones are DNA binding proteins that allow for packaging of the DNA into the nucleus. They are abundantly present across the genome and thus serve as a major site of epigenetic regulation through the use of post-translational modifications (PTMs). Aberrations in histone expression and modifications have been implicated in a variety of human diseases and thus are a major focus of disease etiology studies. A well-established method for studying histones and PTMs is through the chemical derivatization of isolated histones followed by liquid chromatography and mass spectrometry analysis. Using such an approach has allowed for a swath of discoveries to be found, leading to novel therapeutics such as histone deacetylase (HDAC) inhibitors that have already been applied in the clinic. However, with the rapid improvement in instrumentation and data analysis pipelines, it remains important to temporally re-evaluate the established protocols to improve throughput and ensure data quality. Here, we optimized the histone derivatization procedure to increase sample throughput without compromising peptide quantification. An implemented spike-in standard peptide further serves as a quality control to evaluate the propionylation and digestion efficiencies as well as reproducibility in chromatographic retention and separation. Last, the application of various data-independent acquisition (DIA) strategies was explored to ensure low variation between runs. The output of this study is a newly optimized derivatization protocol and mass spectrometry method that maintains high identification and quantification of histone PTMs while increasing sample throughput.