Electrospray ionization mass spectrometry, a leading method for the quantification of biomolecules, is useful for the analysis of posttranslational modifications of proteins. Here we describe a mass spectrometric approach for determining levels of acetylation at individual lysine residues within the amino-terminal tail of histone H4. Because of the high density of acetylatable lysine residues within this short span of amino acids, collision-induced dissociation tandem mass spectrometry was required. In addition, it was necessary to develop an algorithm to determine the fraction of acetylation at specific lysine residues from fragment ions containing more than one lysine residue. This is the first report of direct measurement of endogeneous levels of acetylation at individual lysine residues within the amino-terminal tail of yeast histone H4 and is the first use of tandem mass spectrometry for quantification of peptides containing multiple sites of modification.