Pathogens like viruses, bacteria, protozoa, fungi and helminths have caused and are causing diseases as well as the death of mankind since time immemorial. A proper understanding of the infection mechanisms will aid in designing efficient vaccines, diagnostics and drugs for efficient disease management. When compared to genes, proteins are involved in effector functions of the cell (of the pathogen as well as the host) and play a pivotal role in pathogen entry into host cells, its transmission and disease pathogenesis. Hence, the study of proteins will decipher the intricate communication networks involved in disease pathogenesis and will also elucidate their use as biomarkers in diagnostic and therapeutic scenarios. Thus, this chapter will highlight the proteomic and mass spectrometry-based approaches in comprehending the pathogen interactions with the host at the molecular level as well as biomarker development for use in diagnostics, prognostics and disease management.