Pan-specific anti-acetylated lysine antibody is a valuable tool to detect, validate, and quantify protein lysine acetylation. Compared to site-specific acetyllysine antibodies that are not readily available in most cases, polyclonal pan-specific acetyllysine antibodies can be generated with synthetic antigen of a carrier protein that has its lysine residues chemically acetylated. Here, we describe protocols of synthesizing acetylated ovalbumin (OVA), immunizing rabbits, and characterizing pan-specific polyclonal anti-acetylated lysine antibodies.