MS-GF+ is a new database search tool designed to address the limitations of existing tools in handling diverse spectral data sets. It is sensitive and universal, capable of identifying more peptides than most other tools and performing well across various types of spectra, different MS instrument configurations, and experimental protocols. The tool uses a robust probabilistic model to automatically derive scoring parameters from thousands of peptide-spectrum matches (PSMs) without prior knowledge of the spectrum type. MS-GF+ also computes rigorous E-values using the generating function approach, which significantly improves the number of peptide identifications. Benchmarking against popular tools like Mascot + Percolator shows that MS-GF+ identifies significantly more PSMs for various spectral data sets, including those generated with different fragmentation methods, enzymes, and post-translational modifications. The tool's ability to handle high-precision spectra and novel proteases makes it a versatile and powerful addition to proteomics analysis pipelines.MS-GF+ is a new database search tool designed to address the limitations of existing tools in handling diverse spectral data sets. It is sensitive and universal, capable of identifying more peptides than most other tools and performing well across various types of spectra, different MS instrument configurations, and experimental protocols. The tool uses a robust probabilistic model to automatically derive scoring parameters from thousands of peptide-spectrum matches (PSMs) without prior knowledge of the spectrum type. MS-GF+ also computes rigorous E-values using the generating function approach, which significantly improves the number of peptide identifications. Benchmarking against popular tools like Mascot + Percolator shows that MS-GF+ identifies significantly more PSMs for various spectral data sets, including those generated with different fragmentation methods, enzymes, and post-translational modifications. The tool's ability to handle high-precision spectra and novel proteases makes it a versatile and powerful addition to proteomics analysis pipelines.