Peroxisomes are primarily metabolic organelles with important functions in lipid metabolism, such as fatty acid oxidation and ether phospholipid synthesis (e.g., plasmalogens). Certain viruses, such as human cytomegalovirus (HCMV), hijack organelle functions to facilitate their replication and spread. However, the role of peroxisomes in herpesvirus replication remains elusive. Therefore, we used targeted mass spectrometry to quantify 60 peroxisome proteins through the HCMV infection cycle. We provide two proteomic experiments. The first experiment (raw files labeled as 20180123) is of samples in biological triplicate from uninfected human fibroblasts and infected human fibroblasts at 6, 24, 48, 72, and 120 hours post infection. The second experiment (raw files labeled as 201806) is from uninfected/infected fibroblasts during phosphonoformate (PFA) treatment and fibroblasts infected with UV-treated HCMV.