Aminoacyl‐tRNA synthetases form the protein family that controls the interpretation of the genetic code, with tRNA aminoacylation being the key chemical step during which an amino acid is assigned to a corresponding sequence of nucleic acids. In consequence, aminoacyl‐tRNA synthetases have been studied in their physiological context, in disease states, and as tools for synthetic biology to enable the expansion of the genetic code. Here, we review the fundamentals of aminoacyl‐tRNA synthetase biology and classification, with a focus on mammalian cytoplasmic enzymes. We compile evidence that the localization of aminoacyl‐tRNA synthetases can be critical in health and disease. In addition, we discuss evidence from synthetic biology which made use of the importance of subcellular localization for efficient manipulation of the protein synthesis machinery.This article is categorized under: RNA Processing Translation > Translation Regulation RNA Processing > tRNA Processing RNA Export and Localization > RNA Localization