Introduction: Photoacoustic imaging (PAI) is an emerging technique for the visualization of molecular processes in vitro and in vivo. PAI provides non-invasive images of the absorbed optical energy density at depths of several centimeters with a resolution of ~100 μm.(1) Cancer cells must survive both hypoxic and oxidative stresses as they proliferate to form a tumor mass. PAI is inherently sensitive to oxygen supply, and in turn hypoxic stress, based on endogenous contrast from oxy- and deoxy-hemoglobin. We hypothesize that development of an activatable PAI probe responsive to hydrogen peroxide (H2O2) will enable simultaneous imaging of hypoxic and oxidative stress in tumors in vivo using PAI. Methods: A near-infrared activatable contrast agent was synthesized based on: a heptamethine carbocyanine backbone(2); an H2O2 reactive unit(3),(4) connected via a linker unit; and glucose as a preliminary targeting moiety. Different linker structures between the backbone and H2O2 reactive unit were validated with regard to their ability to induce a change in the optical absorption spectrum of the dye. Optical and optoacoustic spectra were then examined under different environmental conditions (i.a. MeOH, H2O, PBS ± FBS, plasma, ± H2O2). Expected interactions with MeOH and H2O were predicted using density functional theory (DFT). As a first step towards in vivo application, optoacoustic imaging was then performed in tissue mimicking phantoms based on agarose gel containing intralipid and nigrosin black dye. First in vitro studies were performed using the HEK 293 cell line and modifying with concentrations of H2O2 ranging from 50 μM to 2 mM. Results and Discussion: An activatable contrast agent was designed exhibiting an optical absorption peak at 730 nm (in H2O), which shifted by 60 nm to 790 nm (in H2O) after exposure to H2O2 (Fig. 1). Furthermore, the absorbance at 790 nm was found to increase by over 45%. The absorption spectrum was strongly dependent on the solvent, relating to polarity, hydrogen bonding capacity and ion content. The cause of the change in the absorption spectra was clarified by DFT, which showed conformational changes due to solvent interactions. For optoacoustic imaging, the same trend was observed, although peaks were shifted by ~15 nm and broadened. Data acquired in phantoms show promise for future in vivo application. Conclusion: We have synthesized the first activatable contrast agent for PAI that is based on a heptamethine carbocyanine dye and responsive to the presence of H2O2. In the future, we can target this approach using specific molecular markers expressed by cancer cells to study the presence of H2O2 under scenarios including therapeutic response. Future work could provide insight into the role of oxidative stress in cancer, inflammatory processes and other diseases.