Proteomics technologies have produced an abundance of drug targets, which is creating a bottleneck in drug development process. There is an increasing need for better target validation for new drug development and proteomic technologies are contributing to it. Identifying a potential protein drug target within a cell is a major challenge in modern drug discovery; techniques for screening the proteome are, therefore, an important tool. Major difficulties for target identification include the separation of proteins and their detection. These technologies are compared to enable the selection of the one by matching the needs of a particular project. There are prospects for further improvement, and proteomics technologies will form an important addition to the existing genomic and chemical technologies for new target validation. Proteomics is applicable for protein analysis and bioinformatics based analysis gives the comprehensive molecular description of the actual protein component. Bioinformatics is being increasingly used to support target validation by providing functionally predictive information mined from databases and experimental datasets using a variety of computational tools. This review is focused on key technologies for proteomics strategy and their application in protein analysis.