The histone LC-MS/MS data analysis is challenging due to the large number and variety of isobaric histone peptides, and the high dynamic range of histone peptide abundances. We introduce EpiProfile 2.0 to quantify histone post-translational modifications from mass spectrometry data, in which fragment ions are used to determine the retention time of peptides and discriminate isobaric peptides. EpiProfile can automatically recognize and efficiently process both data-dependent and data-independent acquisition runs (DDA and DIA), and high-resolution and low-resolution instrument runs. The proteomics tool was tested on human cells and validated by synthetic histone peptides. EpiProfile is the first automatic program to determine the retention time and discriminate isobaric peptides for histone modifications with DIA MS.