Abstract: An absolute quantification method was developed to quantitatively measure p53 protein using LC-MS/MS for detection of a selective tryptic peptide.A tryptic peptide “VEYLDDR” （m/z 455.2²⁺-681.2¹⁺） was selected of the immunoprecipitation enriched samples of p53 protein following proteolysis with trypsin,and a synthetic peptide “VEYIEDR”（m/z 462.1²⁺-695.2¹⁺）was designed as the internal standard.Quantitativedeterminationof p53 protein changes by the effect of actinomycin D .The lower limit of quantification was 2.2 pmol/mL with the linearity of the standard curve spanned to 112.3 pmol/mL (R²=0.999 3).Both the accuracy (relative error) and the precision (coefficient of variation) of the method were below 15%.This method was successfully applied to determine the absolute amount of p53 in HCT116 cells by the effect of actinomycin D.The p53 expression was increased over time and drug concentration.The developed method could be directly applicable to many current research needs related to p53 protein.