Blast is one of the most important diseases affecting rice cultivation worldwide. The current study employed Two-Dimensional Difference Gel Electrophoresis (2D-DIGE) plus Matrix-assisted laser desorption/ionization time-of-flight-mass spectroscopy (MALDI-TOF MS) and TOF/TOF tandem MS/MS to investigate the differentially regulated proteins in the highly resistant (HR) genotype, FL 127 and highly susceptible (HS) genotype, Rustic against blast pathogen in rice. The differential expression of seventy eight protein spots were observed in 2D-gel analysis of HR and HS genotypes. The comparision of protein expression ratio between HR and HS genotypes showed that twenty protein spots were predominant in differential expression against blast pathogen. Of which, seventeen were upregulated and three were down regulated. Characterization of these proteins using MALDI-TOF (MS) and TOF/TOF tandem MS/MS analysis with protein databases revealed that most of their functions either directly or indirectly related to plant defense and stress response, transcription and photosynthesis. The knowledge on various proteins characterized in this study suggested their possible role in expressing resistance by gentotype FL 127 against P. oryzae. Further, the knowledge generated in this study would be useful in the development of molecular markers for screening blast resistance using molecular biology approaches.