The ribosome is a macromolecular system that has a significant role in synthesizing protein within all living cells. Ribosome in eucaryotic cell consists of two subunits; including small (40S) and large (60S) ribosomal subunits. Moreover, each subunit contains one or more molecules of ribosomal RNA (rRNA) and several ribosomal proteins (r-protein). The purification and isolation of ribosomal proteins from the other cellular organelles are a highly complicated process which needs to do several purification steps to get the desired ribosomal proteins. In this study, the cytosolic ribosomal proteins in the Arabidopsis thaliana cell culture have been purified from other organelles. Also, several types of proteases have been studied to find the best one that hydrolyzes peptide bonds that are used in mass spectrometry protein identification applications. The results have shown that by using double addition of sucrose cushion into the purified sample and using trypsin enzyme, we could purify large number of ribosomal proteins in the sample.