Human cytoplasmic lysyl‐ tRNA synthetase (Lys RS ) is associated within a multi‐aminoacyl‐ tRNA synthetase complex ( MSC ). Within this complex, the p38 component is the scaffold protein that binds the catalytic domain of Lys RS via its N‐terminal region. In addition to its translational function when associated to the MSC , Lys RS is also recruited in nontranslational roles after dissociation from the MSC . The balance between its MSC ‐associated and MSC ‐dissociated states is essential to regulate the functions of Lys RS in cellular homeostasis. With the aim of understanding the rules that govern association of Lys RS in the MSC , we analyzed the protein interfaces between Lys RS and the full‐length version of p38, the scaffold protein of the MSC . In a previous study, the cocrystal structure of Lys RS with a N‐terminal peptide of p38 was reported [Ofir‐Birin Y et al . (2013) Mol Cell 49, 30–42]. In order to identify amino acid residues involved in interaction of the two proteins, the non‐natural, photo‐cross‐linkable amino acid p ‐benzoyl‐ l ‐phenylalanine (Bpa) was incorporated at 27 discrete positions within the catalytic domain of Lys RS . Among the 27 distinct Lys RS mutants, only those with Bpa inserted in place of Lys356 or His364 were cross‐linked with p38. Using mass spectrometry, we unambiguously identified the protein interface of the cross‐linked complex and showed that Lys356 and His364 of Lys RS interact with the peptide from Pro8 to Arg26 in native p38, in agreement with the published cocrystal structure. This interface, which in Lys RS is located on the opposite side of the dimer to the site of interaction with its tRNA substrate, defines the core region of the MSC . The residues identified herein in human Lys RS are not conserved in yeast Lys RS , an enzyme that does not associate within the MSC , and contrast with the residues proposed to be essential for Lys RS :p38 association in the earlier work.