Post-translational modification of proteins is a key regulatory event in many cellular processes. African swine fever virus (ASFV) is a large DNA virus that contains about 150 open reading frames (ORF) which encode for more than 150 polypeptides, most of them without assigned function. Two-dimensional gel electrophoresis (2DE) followed by Post-Source Decay Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (PSD-MALDI-MS) revealed that ASFV protein pE120R, essential for virus transport from assembly sites to plasma membranes, is acetylated at the N-terminal Ala residue during infection. To our knowledge, this is the first acetylated ASFV protein described and this modification might be relevant to ASFV life cycle since many viruses use the acetylation signaling pathway as a primary target for viral proteins after infection.