Metabolic labeling of proteins using classical radioisotope-labeled amino acids has enabled the analysis and function of protein synthesis for many biological processes but cannot be combined with modern high-throughput mass spectrometry analysis. This chapter describes the unbiased identification of a whole de novo synthesized proteome of cultured cells or of a translationally active subcellular fraction of the mammalian brain. This technique relies on the introduction of a small bioorthogonal reactive group by metabolic labeling accomplished by replacing the amino acid methionine by the azide-bearing methionine surrogate azidohomoalanine (AHA) or the amino acid homopropargylglycine (HPG). Subsequently an alkyne- or azide-bearing affinity tag is covalently attached to the group by "click chemistry"-a copper(I)-catalyzed [3+2] azide-alkyne cycloaddition. Affinity tag-labeled proteins can be analyzed in candidate-based approaches by conventional biochemical methods or with high-throughput mass spectrometry.