Publisher Summary The chapter describes a procedure that allows the purification of several aminoacyl-tRNA synthetases simultaneously to such an extent that each individual can be obtained in a homogeneous form by only one further step. The work described is performed mainly with baker's yeast. It is also successfully applied, with only modifications in the method of cell rupture, to the purification of aminoacyl-tRNA synthetases from Neurospora crassa as well as from several bacteria. The strategy of the procedure described depends on the conditions prepurification of aminoacyl-tRNA synthetases as a group by a series of precipitation steps and adsorption to a cation exchanger. Stepwise elution of subfractions containing only few aminoacyl- tRNA synthetases from the cation exchanger and further purification by salting out on Sepharose 4B, which is applicable to proteins in general. Individual enzymes are obtained in a homogeneous state by only one further step optimized for the individual enzyme.