MSFragger is an ultrafast database search tool for peptide identification in mass spectrometry-based proteomics. It has demonstrated excellent performance across a wide range of datasets and applications. MSFragger is suitable for standard shotgun proteomics analyses as well as large datasets (including timsTOF PASEF data), enzyme unconstrained searches (e.g. peptidome), ‘open’ database searches (i.e. precursor mass tolerance set to hundreds of Daltons) for identification of modified peptides, and glycopeptide identification (N-linked and O-linked) with MSFragger Glyco mode.