Lysine acetylation is an important regulatory post-translational modification (PTM) that occurs sub-stoichiometrically, often representing less than 1% of the target protein. This makes studying endogenous protein acetylation extremely challenging. Recent reports suggest that several post-translational modifications (PTMs), including lysine acetylation, play a major role in the regulation the programmed cell death-ligand 1 (PD-L1), a clinically important protein target. An enrichment step is necessary to enable identification of the acetylated species by either antibody or mass spectrometry-based detection methods. This report describes a robust lab protocol for the enrichment and detection of endogenous acetylated PD-L1 protein. A recently developed acetyl lysine affinity matrix was utilized to enrich >90% of acetylated PD-L1 species, from a variety of cell lines, spanning a fourteen-fold range of target protein levels. Western blot analysis, using a highly sensitive PD-L1 antibody and optimized transfer times, was used to determine that the endogenous level of acetylated PD-L1 is in the range of 0.02-0.07% of total PD-L1. As validation, we demonstrate that acetylation levels increase to 0.11-0.17% of total PD-L1 after a 4h treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). The method described here is simple to perform in any lab equipped with tissue culture and western blot equipment.