Abstract: The proteins of K562 cell line were separated by three modes of chromatography, the chromatographic fractions were collected and then detected by MALDI-TOF MS. From comparing the date of those separation modes a conclusion can be made: Using SEC-RPLC separation mode to establish the protein finger print of K562 cell line, more than 120 kinds of proteins from 2 KD to 130 KD can be detected. The detected proteins are the most and the required information is also most comprehensive by using this separation mode. The establishing of this method provides a valid way for clinical study of leukemia.