Post-translational modifications serve as important regulatory elements in modulating the transcriptional activity of the tumor suppressor protein p53. We have previously reported a tandem mass spectrometry-based method (viz. selected ion tracing analysis) that can be applied to the identification of phosphopeptides as well as exact mapping of the phosphorylated residues within. In this study, we describe the application of the same strategy for the identification of p300 acetyltransferase-mediated acetylation sites on p53. Consistent with the previous finding, lysines 370, 372, 373, 381, and 382 were detected by this modified selected ion tracing method as the target sites of p300 in vitro. Moreover, two novel acetylation sites, Lys-292 and Lys-305, were also found. Immunoblotting using anti-acetyl-Lys-305 antibody confirmed this discovery and demonstrated that Lys-305 could be acetylated by p300 both in vitro and in vivo. We also show that an alanine or glutamine substitution at Lys-305 (K305A or K305Q) suppressed the transcriptional activity of p53, whereas an arginine mutation (K305R) increased the transcriptional activity. Thus, p300 may further regulate the transcriptional activity of p53 through a novel acetylation site, Lys-305.