4-Hydroxyderricin is a heat labile bioactive chalcone isolated from the plant Angelica keiskei. It received attention due to its antibiotic potency against several strains of bacteria including pathogens such as Staphylococcus aureus. Despite these promising pharmacological properties, the exact mode of action or the biological targets are still unknown. Here we report the synthesis and the application of a 4-hydroxyderricin probe for activity-based protein profiling (ABPP) in S. aureus. Due to the heat sensitivity of the natural product we utilize a chemical tool for the mild and selective enrichment of labile probe–protein conjugates and report seryl-tRNA synthetase (STS) to be covalently modified by our probe. This modification results in inhibition of the amino acylation of tRNAs catalyzed by S. aureus STS which is an essential enzymatic pathway for bacterial viability.