In this study, a novel substitution method for finding potential protein–protein interactions (PPIs) has been discussed. This newly designed method for analyzing PPI also aids in the comparison of evolutionary distances. The method deals with various data sets, and additionally performs measurable assessment to determine PPIs is introduced. PPIs are biologically relevant and aid in better conceptual framework of phylogenetic profiling. The newly designed framework gives vision to relate the topological properties of the system with evolutionary behavior of datasets. Firstly, this study found that the most conserved protein motifs exist at the roots of the system, whereas newer motifs with mutations have a tendency to dwell on the branches. In-depth functional analysis revealed that the most conserved motifs have high specificity for improved structural procedures and pathway engagements, which may help identify their formative parts in cells. In conclusion, this study demonstrates several important aspects for future studies focusing to enhance phylogenetic profiling systems. This study can also be used effectively to utilize such strategies to develop new biological insights which will further lead to understanding of disease mechanisms. © The Institution of Engineering and Technology 2017.