Protein arginylation-enzymatic addition of the amino acid arginine (Arg) to proteins, mediated by arginyltransferase ATE1, has been discovered in 1963, but is still relatively poorly understood. Studies of arginylation present many technical challenges, which arise from the fact that Arg is a regular amino acid that also incorporates into proteins during translation. Thus, in vitro arginylation needs to be conducted in a strictly ribosome-free system, in highly controlled conditions. Identification of arginylated proteins is currently only possible by high precision mass spectrometry, which relies on very high mass accuracy of the instruments, specific ionization patterns during mass fragmentation, as well as multiple stringent steps of automated and manual validation. Below we describe the methods of in vitro arginylation and mass spectrometry analysis of arginylated proteins, developed by our groups during the last 15 years.