There are two methods used to calculate data for protein quantification using LC-MS/MS with TMT labeling. Spectra-sum method is calculated as a sum of the intensities of TMT report ions in spectra level. In other hand, Spectra-ratio method is calculated as a median of the ratio intensities with the global reference ion. In order to find a best quantification method, we prepared Hela standard peptides with TMT-6-plex labeling of difference protein content ratio as 0.5:0.5:1.0:1.0:2.0:2.0. First, we identified 3,684 proteins with FDR < 1% as spectra and global protein level using Integrated Proteomic Pipeline and ProteinInferencer. Of 30 replicated experiment of day by day, 16,384 is median number of identified spectra. We calculated the ratio value between the different protein content samples using the TMT reporter ions. As a result of spectra-sum method, the mean value of the ratio of number of proteins with CV < 30% was 0.865, and its standard deviation (SD) was low (0.07). The other hand of spectra-ratio method, the mean value was 0.45 and SD was high (0.11). It is indicated that spectra-sum method is more accurate quantification, where 87% of proteins could be quantifiable.