Tandem mass spectrometry of peptides is essential to protein identification.  However, weak signals and incomplete sequence ions in the mass spectra prevent de novo sequencing of peptides and result in false positives in automatic protein identification in proteomics.  We investigated ion fragmentation in a tandem mode of in-source fragmentation (ISF) and parallel collision induced dissociation (pCID) in a collision cell. In such an approach, all the peptide ions are subjected to the two sequential fragmentations in a tandem mass spectrometer without precursor selection ─ a method that can be called shotgun² collision-induced dissociation mass spectrometry. Solutions of [Glu¹]-fibrinopeptide B (GluFib), TNF-α (46-65), Leu-Enkephalin-Lys and Dynorphin A (1-6) in 50:50 methanol:water (v/v) and 0.2% formic acid were infused into a tandem mass spectrometer (QTOF Micro, Waters) by a syringe pump.  The mass spectrometer was operated in ESI-TOF-MS mode, allowing all the ions passing the first quadrupole mass analyzer to enter the collision cell.  Resulting mass spectra showed increased sequence coverage and increased intensities of y-ions, in comparison with mass spectra obtained via serial MS/MS experiments. The ratios of the absolute intensities of y-ions from shotgun² CID to those from serial MS/MS showed two to ten times increases in sensitivity for the y-ions. These results suggest the potential proteomics applications of the shotgun² CID experiments on commercial tandem mass spectrometers.