The acetylation of proteins at specific lysine residues by acetyltransferase enzymes has emerged as a posttranslational modification of high biological impact. While lysine acetylation in histone proteins is an integral part of the histone code the acetylation of a multitude of non-histone proteins was recently recognized as a regulatory signal in many cellular processes. New substrates of acetyltransferase enzymes are continuously identified and the analysis of acetylation sites in proteins is increasingly performed by mass spectrometry. However, the characterization of lysine acetylation in proteins using mass spectrometric techniques has some limitations and pitfalls. Especially the non-enzymatic cysteine acetylation can result in false-positive identification of acetylated proteins. Here, we demonstrate the application of various mass spectrometric techniques such as matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry for the analysis of protein acetylation. We describe diverse combinations of biochemical methods useful to map the acetylation sites in proteins and discuss their advantages and limitations. As example, we present a detailed analysis of the acetylation of the HIV-1 transactivator of transcription (Tat) protein which is known to be acetylated in vivo by the acetyltransferases p300 and PCAF.