Photodynamic treatment (PDT) is an emerging therapeutic procedure for the management of cancer, based on the use of photosensitizers, compounds that generate highly reactive oxygen species (ROS) on irradiation with visible light. The ROS generated may oxidize a variety of biomolecules within the cell, loaded with a photosensitizer. The high reactivity of these ROS restricts their radius of action to 5–20 nm from the site of their generation. We studied oxidation of intracellular proteins during PDT using the ROS-sensitive probe acetyl-tyramine-fluorescein (acetylTyr-Fluo). This probe labels cellular proteins, which become oxidized at tyrosine residues under the conditions of oxidative stress in a reaction similar to dityrosine formation. The fluorescein-labeled proteins can be visualized after gel electrophoresis and subsequent Western blotting using the antibody against fluorescein. We found that PDT of rat or human fibroblasts, loaded with the photosensitizer Hypocrellin A, resulted in labeling of a set of intracellular proteins that was different from that observed on treatment of the cells with H2O2. This difference in labeling patterns was confirmed by 2D electrophoresis, showing that a limited, yet distinctly different, set of proteins is oxidized under either condition of oxidative stress. By matching the Western blot with the silver-stained protein map, we infer that α-tubulin and β-tubulin are targets of PDT-induced protein oxidation. H2O2 treatment resulted in labeling of endoplasmic reticulum proteins. Under conditions in which the extent of protein oxidation was comparable, PDT caused massive apoptosis, whereas H2O2 treatment had no effect on cell survival. This suggests that the oxidative stress generated by PDT with Hypocrellin A activates apoptotic pathways, which are insensitive to H2O2 treatment. We hypothesize that the pattern of protein oxidation observed with Hypocrellin A reflects the intracellular localization of the photosensitizer. The application of acetylTyr-Fluo may be useful for characterizing protein targets of oxidation by PDT with various photosensitizers.