This chapter describes procedures for purification of μ-calpain, m-calpain, and calpastatin simultaneously from a single tissue sample. The procedures and reagents described apply equally well to purification of any one or two of these three proteins from an appropriate tissue with a corresponding decrease in effort. The series of steps and columns that can be employed permits many variations, but at the outset there are some important initial choices to be made concerning tissue source, means of homogenization, and initial steps of fractionation.