Histidine phosphorylation (pHis), which plays a key role in signal transduction in bacteria and lower eukaryotes, has been shown to be involved in tumorigenesis. Due to its chemical instability, substoichiometric properties, and lack of specific enrichment reagents, there is a lack of approaches for specific and unbiased enrichment of pHis-proteins/peptides. In this study, an integrated strategy was established and evaluated as an unbiased tool for exploring the histidine phosphoproteome. First, taking advantage of the lower charge states of pHis-peptides versus the non-modified naked peptides at weak acid solution (∼pH 2.7), strong cation exchange (SCX) chromatography was used to differentiate modified and non-modified naked peptides. Furthermore, selective enrichment of the pHis-peptide was performed by applying Cu-IDA beads enrichment. Finally, stable isotope dimethyl labeling was introduced to guarantee high-confidence assignment of pHis-peptides. Using this integrated strategy, 563 different pHis-peptides (H = 1) in 385 proteins were identified from HeLa lysates. Motif analysis revealed that pHis prefers hydrophobic amino acids and has the consensus motif-HxxK, which covered the reports from different approaches. Thus, our method may provide an unbiased and effective tool to reveal histidine phosphoproteome and to study the biological process and function of histidine phosphorylation.