We report a proximity labeling-based orthogonal trap approach for investigation of HDAC8 substrates in living cells. In this strategy, the genetic fusions that coupled the engineering enzyme APEX2 with HDAC8 are designed to trap substrate proteins in cellular context, by which they contribute to the covalent tagging of the transient neighboring binders. The tagged proteins are then employed with an integrated orthogonal enrichment strategy that allows for direct identification of acetylated sites on targets.