Proteins provide the verbs to biology, and proteomics provides the nouns for their analytical and discovery-driven studies. The term proteomics was coined in the 1990s and deals with the protein complement of the genome-the proteome. Following the classical proteomics era, the development of new mass spectrometric methods for peptide analysis permitted the identification of proteins in peptide mixtures obtained by proteolytic digestion of complex samples, e.g., shotgun proteomics. Since its introduction, shotgun proteomics became the standard technique for the analysis of protein hydrolyzates in a high-throughput way. In this chapter, we provide a survey in shotgun proteomics highlighting instruments and techniques used in modern second and third proteomics generation.