Hypothetical proteins (HPs) are the proteins predicted to be expressed from an open reading frame, making a substantial fraction of proteomes in both prokaryotes and eukaryotes. Genome projects have led to the identification of many therapeutic targets, the putative function of the protein, and their interactions. In this review we enlist various methods linking annotation to structural and functional prediction of HPs that assist in the discovery of new structures and functions serving as markers and pharmacological targets for drug designing, discovery, and screening. Further we give an overview of how mass spectrometry as an analytical technique is used to validate protein characterisation. We discuss how microarrays and protein expression profiles help understanding the biological systems through a systems-wide study of proteins and their interactions with other proteins and non-proteinaceous molecules to control complex processes in cells. Finally, we articulate challenges on how next generation sequencing methods have accelerated multiple areas of genomics with special focus on uncharacterized proteins.