Enhanced in-source fragmentation/annotation (EISA) has recently been shown to produce fragment ions that match tandem mass spectrometry data across a wide range of small molecules. EISA has been developed to facilitate data-dependent acquisition (DDA), data-independent acquisiton (DIA), and multiple-reaction monitoring (MRM), enabling molecular identifications in untargeted metabolomics and targeted quantitative single-quadrupole MRM (Q-MRM) analyses. Here, EISA has been applied to peptide-based proteomic analysis using optimized in-source fragmentation to generate fragmentation patterns for a mixture of 38 peptides, which were comparable to the b- and y-type fragment ions typically observed in tandem MS experiments. The optimal in-source fragmentation conditions at which high-abundance peptide fragments and precursor ions coexist were compared with automated data-dependent acquisition (DDA) in the same quadrupole time-of-flight (QTOF-MS) mass spectrometer, generating a significantly higher fragment percentage of peptides from both singly and doubly charged b- and y-type fragment (b+, y+, b2+, and y2+) ions. Higher fragment percentages were also observed for these fragment ion series over linear ion trap instrumentation. An XCMS-EISA annotation/deconvolution program was developed, making use of the retention time and peak shape continuity between precursor fragment ions, to perform automated proteomic data analysis on the enhanced in-source fragments. Post-translational modification (PTM) characterization on peptides was demonstrated with EISA, producing fragment ions corresponding to a neutral loss of phosphoric acid with greater intensity than observed with DDA on a QTOF-MS. Moreover, Q-MRM demonstrated the ability to use EISA for peptide quantification. The availability of more sophisticated in-source fragmentation informatics, beyond XCMS-EISA, will further enable EISA for sensitive autonomous identification and Q-MRM quantitative analyses in proteomics.