Canonically, tRNA synthetases charge tRNA. However, the lysyl‐tRNA synthetase paralog EpmA catalyzes the attachment of (R)‐β‐lysine to the ε‐amino group of lysine 34 of the translation elongation factor P (EF‐P) in Escherichia coli. This modification is essential for EF‐P‐mediated translational rescue of ribosomes stalled at consecutive prolines. In this study, we determined the kinetics of EpmA and its variant EpmA_A298G to catalyze the post‐translational modification of K34 in EF‐P with eight noncanonical substrates. In addition, acetylated EF‐P was generated using an amber suppression system. The impact of these synthetically modified EF‐P variants on in vitro translation of a polyproline‐containing NanoLuc luciferase reporter was analyzed. Our results show that natural (R)‐β‐lysylation was more effective in rescuing stalled ribosomes than any other synthetic modification tested. Thus, our work not only provides new biochemical insights into the function of EF‐P, but also opens a new route to post‐translationally modify proteins using EpmA.