Protein phosphorylation is one of the post-translational modifications. Obtaining quantitative information on post-translational modification patterns of proteins is crucial to understanding the signaling and regulatory processes in many diseases. Despite many studies to quantify phosphorylation levels in biological systems, it is still difficult to measure the absolute phosphorylation level due to the lack of reliable quantitative methods. In this study, we developed the quantitative assay platform for multi-phosphorylated peptide consisting of three steps using multiple reaction monitoring (MRM). We first selected and synthesized single & multi-phosphopeptide to be phosphorylated by a serine, threonine and tyrosine; DRVS*IHPF, DRVY*IHPF, DRVT*IHPF, GADDSYY*TAR, GADDSY*Y*TAR, TRDIY*ETDY*Y*RK (* means phosphorylation). In the second step, we analyzed the mixture of phospho/non-phosphopeptides using Q-Exactive mass spectrometry to confirm the synthetic peptides and make a spectral library. In order to find the best method of enrichment efficiency in four different enrichment techniques; Fe-NTA (Thermo), TiO2 (Thermo), magnetic TiO2 (Pierce), TiO2 Mag Sepharose (GE Healthcare), we compared the efficiency of phosphopeptide enrichment. The third step was to assess whether our assay platform showed the quantitative accuracy, stability and reproducibility in measuring the phosphorylation level; Response curves were generated, reproducibility was verified, and six phosphopeptides were evaluated for selectivity. Moreover, we followed the “CPTAC Assay Guidelines”, which provides a list of experiments that will help users obtain more reliable data for more accurate and stable quantitative assays. In conclusion, our developed the quantitative assay platform can be applied to more complicated samples such as tissue or cell lysates to analyze new phosphorylation sites and more phosphorylated peptides.