The article presents a method for absolute quantification (AQUA) of proteins and their post-translational modifications using tandem mass spectrometry. The AQUA strategy involves synthesizing peptides with stable isotopes as internal standards, which can also be covalently modified to mimic natural post-translational modifications. These synthetic peptides are used to quantitatively measure the absolute levels of proteins and modified proteins after proteolysis. The method was validated using horse heart myoglobin in a yeast background, low-abundance yeast proteins involved in gene silencing, and human separase Ser-1126 phosphorylation. The AQUA strategy was also used to identify kinases capable of phosphorylating Ser-1501 of human separase in an in vitro kinase assay. The AQUA method provides a focused approach for studying the dynamically changing proteome and can be applied to various biological systems.The article presents a method for absolute quantification (AQUA) of proteins and their post-translational modifications using tandem mass spectrometry. The AQUA strategy involves synthesizing peptides with stable isotopes as internal standards, which can also be covalently modified to mimic natural post-translational modifications. These synthetic peptides are used to quantitatively measure the absolute levels of proteins and modified proteins after proteolysis. The method was validated using horse heart myoglobin in a yeast background, low-abundance yeast proteins involved in gene silencing, and human separase Ser-1126 phosphorylation. The AQUA strategy was also used to identify kinases capable of phosphorylating Ser-1501 of human separase in an in vitro kinase assay. The AQUA method provides a focused approach for studying the dynamically changing proteome and can be applied to various biological systems.