Mycobacterium tuberculosis, the causative agent of tuberculosis accounts for 1.5 million annual deaths worldwide. The two well-characterized strains of the parental H37 strain namely, H37Ra and H37Rv show different pathogenic phenotypes. In order to identify factors that are responsible for virulence, we compared the proteome and the phosphoproteome profiles of virulent (H37Rv) and virulence attenuated (H37Ra) strains of M. tuberculosis. Quantitative proteomic analysis resulted in the identification and quantitation of 2,709 proteins and 505 phosphosites. Comparative analysis revealed over 5-fold overexpression of several proteins associated with virulence. Our data indicates that there are definable molecular differences between H37Rv and H37Ra strains at both the proteome and phosphoproteome levels which may explain the virulence and phenotypic differences.