SEQUEST is the most widely used software tool for identifying proteins in complex mixtures. It is a mature, robust program that identifies peptides directly from uninterpreted tandem mass spectra, thus making large-scale proteomic studies possible. Thermo Electron's TurboSEQUEST provides a Windows-based graphical user interface for running SEQUEST and interpreting results. The protocol in this unit describes the basic steps involved in processing mass spectrometric data and analyzing results using TurboSEQUEST. It also provides an overview of the SEQUEST algorithm and a discussion of data filtering methods, critical issues in data interpretation, and available resources that can facilitate proper interpretation of SEQUEST results.