Chromosomes are supermolecules that contain most of the DNA within a cell and are visible under optical and electron microscopes. Although they were observed at the earliest stage of genetics, their fundamental structure is not yet understood. The reasons for this are debated among researchers; however, it is clear that the accumulation of metaphase chromosomes for their biochemical analysis has been a significant challenge. In this chapter, a method is described for accumulating and preparing human metaphase chromosomes in sufficient amount to perform their proteome analysis. Preparation and separation methods of chromosome proteins are described, followed by a protocol for their identification by mass spectrometry.