Proteins function at the right time and at the right place for various cells to function properly. Changes in gene instructions causes severe medical problems. These changes can be identified by protein alignment and its substitution groups. Protein alignments reflects various properties, which aid in identifying the cause of the problems. This paper introduces a method for theoretical identification of amino acid substitution groups. This method approaches to view amino acid substitution as a pair wise phenomenon and characterizes it using binary matrix. Amino acids satisfy various properties. It cannot be decided which property is most important in classifying and determining protein structure. Based on the existing method of multi property based classification, the proposed binary matrix is created to identify amino acid pairs so that their pair wise property score is atleast 50 percent. © 2015, Sphinx Knowledge House. All right reserved.