Whole genome sequencing technology provided expensive information for the identifi cation of new therapeutic targets in pathogens over and above human genome. Subtractive genomic approach is extremely informative technique to identify the potential targets, which are expected to be essential genes or proteins in pathogen but absent in host, which can be used as drug target. Besides it uncharacterized proteins, which are present on the exposed surface of pathogen, may also be consider as drug target. In present study subtractive genomic approach has been used to identify therapeutic target in Mycoplasma pneumoniae, which is atypical pneumonia causing pathogen in human. The subsequent analysis revealed that 732 genes were coding 693 proteins in M. pneumoniae out of which 71 proteins were duplicate and 220 proteins were found essential nonhuman homolog. Further analysis of these non human homologous proteins predicted that 27 essential proteins were involved in unique metabolic pathways of Mycoplasma pneumoniae. Therefore these 27 essential proteins may serve as therapeutics target. Protein localization predictions of 220 essential proteins were exposing that 12 proteins were present on the exposed surface of pathogen. These exposed surface proteins could be the possible drug targets as well.