We applied an ISG15-GlyGly peptidomics approach to HeLa cells infected with monkeypox virus (MPXV). Cells were lysed in 5% SDS and processed using S-Trap columns. Following trypsin digestion, GlyGly-modified peptides were enriched and subsequently identified and quantified by LC–MS/MS. In addition, a shotgun proteomics analysis was performed on the same samples prior to GlyGly-enrichment.