The clinical use of valproic acid (VA) as an antiepileptic drug is associated with adverse effects such as hepatotoxicity and encephalopathy. These potential complications may be caused by reactive oxygen species (ROS) produced during the metabolic processing of VA. In this study, we used screening to identify cellular protein markers of VA-induced oxidative stress in human liver cells. To confirm that the protein modifications were induced by VA, nano-ultra-performance liquid chromatography (nanoUPLC) coupled with tandem mass spectrometry (MS/MS) was used for structural identification. Protein modifications induced by oxidative stress were found to involve crotonaldehyde, 4-hydroxynonenal, an oxygen atom, nitric oxide, and the nitro and acetyl groups. The data on the above-mentioned protein modifications may serve as indicators of apoptosis, oxidative stress, or other adverse reactions induced by the clinical use of VA.