Most pharmaceutical peptides are supplied as acetate salts and the relative amount of acetate to peptide in the final product is one quality criterion required by regulatory agencies to approve the product for clinical use. The objective of the present study was to develop a validated LC-MS/MS method that allows the quantitative determination of the acetate content in pharmaceutical peptide preparations and simultaneous monitoring and collection of qualitative and quantitative information on the peptide during manufacture and in the final product. The method uses reversed phase C18-chromatography to elute the acetate ions under acidic conditions, pH 3, followed by post-column infusion of ammonium hydroxide 0.6 M, methanolic solution (30:70) at a rate of 0.5 mL/hr. The acetate ions were monitored in negative polarity mass spectrometry by pseudo multiple reaction monitoring (pseudo MRM) against 1, 2- 13C labelled acetate, the internal standard used in the method. The method was linear for acetate concentrations between 0.4 and 25 μg/mL with a coefficient of determination (r2) equal to 0.9999. The minimum level of detection and minimum level of quantification were at 0.06 μg/mL and 0.18 μg/mL respectively. Accuracy of the method was judged by determining the acetate content in a commercial product of the peptide pharmaceutical tetracosactide (TCS) and parallel comparison to the amounts determined by a reversed phase HPLC method with detection at a wavelength of 210 nm. The amounts determined by the two methods were in agreement with a RSD that was less than 2%. Additional confirmation of method accuracy was determined by spiking the pharmaceutical peptide with varying amounts of acetate. The recoveries ranged on average between 101 and 102% for the spiked amounts. Accuracy was also determined by calculating the percentage relative error of the predicted to actual acetate concentration in quality controls and was determined to be less than 5%. The LC-MS/MS method was precise with an intra- and inter-day RSD of less than 5%. The standard solutions were stable for at least one month when kept frozen at −80 °C with no loss in response and an inter-day RSD of less than 5%. The method was applied to quantify the acetate content in the clinically available product of TCS and to simultaneously evaluate the average peptide molecular weight and detect known impurities by switching from negative polarity MRM analysis to positive polarity MS analysis following the elution of the acetate peak. The method reported herein should corroborate quantitative determinations of the acetate content in pharmaceuticals by the traditional compendial HPLC method.