Stable isotope labeling by amino acids in cell culture (SILAC) is an in vivo metabolic labeling technique based on the abundance ratio of mass spectrometry. Combined with different methods of proteins affinity enrichment, SILAC can obtain sensitive, accurate and quantitative information of proteins. This article reviewed the history, rationale, characteristics and application of SILAC, with special focus on its role in studying protein interactions with small molecules, quantitative proteomics and post-translational modification of proteins.