A new method for absolute quantification of proteins using LCMS$^{E}$ is described. This method relies on the consistent average MS signal response for the three most intense tryptic peptides per mole of protein, which is used to calculate a universal signal response factor. This factor is the same for all proteins tested. The method was validated using six exogenous proteins of varying concentrations, with a relative error of less than ±15%. The average MS signal responses of the three most intense peptides from each protein were plotted against their calculated concentrations, resulting in a linear relationship with an R² value of 0.9939. The method was applied to determine the absolute concentration of 11 common serum proteins, which were compared with known values. Additionally, the method was used to determine the stoichiometry of proteins in an Escherichia coli lysate. The study highlights the importance of absolute quantification in understanding proteome variability and cellular responses. The method provides a means to determine the absolute concentration of proteins in complex mixtures without the need for external standards or radiolabeling. The method uses the consistent signal response of the three most intense tryptic peptides to calculate the absolute concentration of proteins. The results show that the average MS signal response for these peptides is constant per unit quantity of protein, allowing for accurate quantification. The method was validated using a variety of samples, including human serum and E. coli proteins, demonstrating its effectiveness in complex matrices. The study concludes that the method is suitable for determining the absolute concentration of proteins in both simple and complex mixtures, providing a valuable tool for proteomics research.A new method for absolute quantification of proteins using LCMS$^{E}$ is described. This method relies on the consistent average MS signal response for the three most intense tryptic peptides per mole of protein, which is used to calculate a universal signal response factor. This factor is the same for all proteins tested. The method was validated using six exogenous proteins of varying concentrations, with a relative error of less than ±15%. The average MS signal responses of the three most intense peptides from each protein were plotted against their calculated concentrations, resulting in a linear relationship with an R² value of 0.9939. The method was applied to determine the absolute concentration of 11 common serum proteins, which were compared with known values. Additionally, the method was used to determine the stoichiometry of proteins in an Escherichia coli lysate. The study highlights the importance of absolute quantification in understanding proteome variability and cellular responses. The method provides a means to determine the absolute concentration of proteins in complex mixtures without the need for external standards or radiolabeling. The method uses the consistent signal response of the three most intense tryptic peptides to calculate the absolute concentration of proteins. The results show that the average MS signal response for these peptides is constant per unit quantity of protein, allowing for accurate quantification. The method was validated using a variety of samples, including human serum and E. coli proteins, demonstrating its effectiveness in complex matrices. The study concludes that the method is suitable for determining the absolute concentration of proteins in both simple and complex mixtures, providing a valuable tool for proteomics research.