Two novel compounds were identified as Naa50 binders/inhibitors using DNA-encoded technology screening. Biophysical and biochemical data as well as cocrystal structures were obtained for both compounds (<b>3a</b> and <b>4a</b>) to understand their mechanism of action. These data were also used to rationalize the binding affinity differences observed between the two compounds and a MLGP peptide-containing substrate. Cellular target engagement experiments further confirm the Naa50 binding of <b>4a</b> and demonstrate its selectivity toward related enzymes (Naa10 and Naa60). Additional analogs of inhibitor <b>4a</b> were also evaluated to study the binding mode observed in the cocrystal structures.