Mass spectrometry (MS) measures the mass-to-charge ratio of ions to identify and quantify molecules in simple and complex mixtures. MS has become invaluable across a broad range of fields and applications, including proteomics. The development of high-throughput and quantitative MS proteomics workflows within the last two decades has expanded the scope of what we know about protein structure, function, modification and global protein dynamics. This overview outlines the role of mass spectrometry in the field of proteomics and reviews MS methodology and instrumentation and touches on sample preparation and liquid chromatography-based separation prior to MS analysis.