In most naturally occurring glycoproteins, pools of glycans are attached to one or more glycosylation sites. The analysis of each individual site is challenging and requires the analysis of glycopeptides. For this, quadrupole time of flight (QTOF) mass spectrometers (MS) with high mass accuracy, fast duty cycles, and high m/z range are highly suited due to the usability of multiple collision energies. In this study we systematically investigated the optimum collision energies required for fragmentation of the glycan and the peptide parts of glycopeptides, and present a software-supported approach for data interpretation. For the initial studies of fragmentation energy, synthetic N-glycopeptides were analysed on a QTOF MS instrument (impact II) with CaptiveSpray nanoBooster^tm (Bruker Daltonics). For further testing, tryptically digested standard glycoproteins (fetuin, antibodies) were separated by nano LC before MS analysis using systematically varied collision energies. Glycopeptide spectra were detected and the peptide masses were determined automatically (ProteinScape 4.0, Bruker Daltonics). Glycan structures were identified using the integrated GlycoQuest search engine, and for peptide identification Mascot (Matrix Sciences) was used. The energies required for optimal glycan fragmentation were found to be clearly below the ones necessary for the peptide part. Collision energy stepping allowed identification of both peptide and glycan moieties from a single MSMS spectrum in ProteinScape 4.0. In summary, the optimized parameters were successfully applied on digested monoclonal antibodies and glycoprotein mixtures, which resulted in the identification of complete N- and O-glycopeptides. This improved methodology is particularly useful in the fields of glycoproteomics research as well as biopharmaceutical development and quality control.