The history of mass spectrometry started at the beginning of the 20th century. Since then, it has undergone an enormous expansion in terms of improvements to technical and methodological innovations, which has benefited the growth of its application in chemistry, biochemistry, pharmacology and medicine. Mass spectrometry is an analytical technique that allows molecular mass determination and structural elucidation for both known and unknown molecules. This chapter presents a general introduction to mass spectrometry instrumentation and the basic ‘know-how’ of operating principles of ionization systems and mass analysers for the study of biomolecules.