The main goal of the present study is to develop a method to recognize and identify endogenous intrachain disulfide bonded peptide, which are rarely sequenced in current peptidomics studies. In order to achieve highly efficient detection of these peptides in a neuropeptidome analysis, we alkylated the peptides, mined the raw mass spectrometry data, and then recognized the candidates of untreated disulfide bonded peptides from unalkylated peptide extracts. After removing more than 90% features, targeted electron transfer dissociation fragmentation was performed for detecting and fragmenting disulfide bonded peptides, and even most of them were present in low abundance in the original sample. Diverse endogenous disulfide bonded peptides were then detected and sequenced, opening up new perspectives for comprehensively understanding the response of a neuropeptidome.