Proteins are the major functional unit that dictates the structure and function of a cell, tissue or organism. The term proteome refers to the pool of proteins coded by the whole genome. Significant efforts towards deciphering the secrets of the human genome in the next-generation genomics era, with high-throughput technologies, failed to provide functional relevance, as gene expressions do not necessarily correlate with protein expression. Therefore, proteomics has emerged as an independent stream of biological research for the large-scale comprehensive systematic study of the proteome of a given cell, organism or condition. Mass spectrometry has proven to be central to proteomics-based studies as it not only helps in the identification of proteins but has also been successfully used for quantification. Initially gel-based (both 1 and 2 dimensions) proteomics played a significant role in the identification and quantification of proteins in a given experimental condition. However gel-based proteomics, has its own limitations and is a cumbersome process. The evolution of liquid chromatography and mass spectrometry has played a pivotal role for the development of proteomics and they are now routinely used for qualitative and quantitative analyses. One of the most important aspects of proteomics is data analysis. A large amount of high-throughput and multidimensional data generated from proteomics experiments needs to be analyzed with profound accuracy. Therefore understanding the principles of the various analysis tools is also essential to making sense of the complex proteomics data. Applications of these advanced sophisticated tools have helped enormously in deciphering critical nodes in biological research. In this chapter, we aim to provide a snapshot of “proteomics”, from sample collection to data analysis, and, most importantly, its application towards solving critical biological questions.