A prominent posttranslational modification associated with oxidative stress is 4-hydroxynonenal (HNE) protein adduction. Numerous studies have assessed oxidative stress but HNE protein modification has not been determined in any marine organism. LCMS proteomics was used to identify 1052 HNE adducted proteins in the cosmopolitan tunicate Botryllus schlosseri sourced from field and laboratory populations. Adducted amino acid residues were ascertained for 1849 modified sites, of which 1195 had a maximum localization score. Most HNE modifications were at less reactive lysines (rather than more reactive cysteines). HNE modification on most sites was high as corresponding unmodified peptides were undetectable. These observations suggest that Botryllus experiences high intracellular reactive oxygen species levels, resulting in substantial lipid peroxidation. Botryllus proteins with HNE adducts show enrichment in mitochondrial, proteostasis, and cytoskeletal functions. A DIA assay library was constructed to quantify HNE adduction at 72 sites across 60 proteins and reveal significantly higher HNE adduction in laboratory versus field populations. Based on the function of HNE adducted Botryllus proteins, we propose that redox signaling contributes to regulating senescence and cytoskeleton dynamics during Botryllus development and physiology. Overall, HNE modified proteins represent promising biomarkers of oxidative stress in tunicates and other aquatic species inhabiting variable environments.