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Recent advances in proteome informatics have led to an explosion in tools to analyze mass spectrometry data. These tools operate across the analysis pipeline doing everything from assessing quality control to matching peptides to spectra to quantification. Unfortunately, the vast majority of these tools are not able to operate directly on the proprietary formats generated by the diverse mass spectrometers. Consequently, the first step in many protocols is the conversion of data from vendor-specific binary files to open-format files. This protocol details the use of ProteoWizard’s msConvert and msConvertGUI software for this conversion, taking format features, coding options, and vendor particularities into account. We specifically describe the various options available when doing conversions and the implications of each option.