Histone modifications play an important role in regulating transcriptional gene expression and chromatin processes in eukaryotes. Increasing researches proved that aberrant post-translational modiﬁcations (PTMs) of histones is associated with many diseases. However, MS-based identification and quantification of histone PTMs is still challenging. Although classic chemical derivatization in conjunction with trypsin digestion protocol is a widely used in histone PTMs analysis in bottom-up strategy, several side reactions have been observed in practice. In this work, outer membrane protease T OmpT was utilized as a protease for direct histone proteolysis and generated convenient histone H3 peptide lengths for retention on reversed-phase chromatography. The powerful and unique tolerance of OmpT for modified lysine and arginine was demonstrated and quantitatively described for the first time, making it useful for detecting naturally modifications. Using the optimized digestion conditions, we succeeded to identified most of abundant modifications on histone from HEK293T cells. Additionally, histone H3 PTMs were quantitatively profiled in KMS11 cell line with selective knock out of translocated NSD2 allele (TKO) and the original parental KMS11 cell lines (PAR), revealed the NSD2 was of high specificity on H3K36 dimethylation. Histone chemical derivatizations are not required in our strategy, showing a remarkable strength over the conventional trypsin-based workflow.