A method for absolute quantification of a first polypeptide in a sample comprising a plurality of polypeptides comprising the first polypeptide and a second polypeptide, wherein the first polypeptide is at least 10-fold lower in abundance than the second polypeptide and the second polypeptide has a known concentration in the sample, the method comprising: (a) obtaining MS signals of ions of peptide products of the plurality of polypeptides, wherein said MS signals of ions of the peptide products are obtained by analyzing the peptide products of the plurality of polypeptides using a liquid chromatography/mass spectrometry (LC/MS) technique, wherein MS signals of the peptide products are obtained for each of a plurality of sample loading quantities comprising a first sample loading quantity and a second sample loading quantity, and wherein the first sample loading quantity is greater than the second sample loading quantity; (b) calculating the average or sum of an MS signal for: (i) a top set of n number of qualified ions of peptide products of the first polypeptide with the highest MS signals at the first sample loading quantity; (ii) a top set of n number of qualified ions of peptide products of the second polypeptide with the highest MS signals at the second sample loading quantity; (iii) a middle set of m number of qualified ions of peptide products of the second polypeptide at the first sample loading quantity; and (iv) the middle set of qualified ions of peptide products of the second polypeptide at the second sample loading quantity, wherein A, B, C, and D are all calculated using the average or all calculated using the sum of the MS signal; and (c) determining an absolute quantity of the first polypeptide in the first sample loading quantity based on the following formula: A / C * mole of the second polypeptide at the first loading quantity * D / B. (a) obtaining MS signals of ions of peptide products of the plurality of polypeptides, wherein said MS signals of ions of the peptide products are obtained by analyzing the peptide products of the plurality of polypeptides using a liquid chromatography/mass spectrometry (LC/MS) technique, wherein MS signals of the peptide products are obtained for each of a plurality of sample loading quantities comprising a first sample loading quantity and a second sample loading quantity, and wherein the first sample loading quantity is greater than the second sample loading quantity; wherein said MS signals of ions of the peptide products are obtained by analyzing the peptide products of the plurality of polypeptides using a liquid chromatography/mass spectrometry (LC/MS) technique, wherein MS signals of the peptide products are obtained for each of a plurality of sample loading quantities comprising a first sample loading quantity and a second sample loading quantity, and wherein the first sample loading quantity is greater than the second sample loading quantity; (b) calculating the average or sum of an MS signal for: (i) a top set of n number of qualified ions of peptide products of the first polypeptide with the highest MS signals at the first sample loading quantity; (ii) a top set of n number of qualified ions of peptide products of the second polypeptide with the highest MS signals at the second sample loading quantity; (iii) a middle set of m number of qualified ions of peptide products of the second polypeptide at the first sample loading quantity; and (iv) the middle set of qualified ions of peptide products of the second polypeptide at the second sample loading quantity, wherein A, B, C, and D are all calculated using the average or all calculated using the sum of the MS signal; and (i) a top set of n number of qualified ions of peptide products of the first polypeptide with the highest MS signals at the first sample loading quantity; (ii) a top set of n number of qualified ions of peptide products of the second polypeptide with the highest MS signals at the second sample loading quantity; (iii) a middle set of m number of qualified ions of peptide products of the second polypeptide at the first sample loading quantity; and (iv) the middle set of qualified ions of peptide products of the second polypeptide at the second sample loading quantity, (c) determining an absolute quantity of the first polypeptide in the first sample loading quantity based on the following formula: