<p>The development of high-performance photoacoustic (PA) probes that can monitor disease biomarkers in deep-tissue has the potential to replace invasive medical procedures such as a biopsy. However, such probes must be highly optimized for <i>in vivo</i> performance and exhibit an exceptional safety profile. In this study, we have developed PACu-1, the first PA probe designed for biopsy-free assessment (BFA) of hepatic Cu via photoacoustic imaging. PACu-1 features a Cu(I)-responsive trigger appended to an aza-BODIPY dye platform that has been optimized for ratiometric sensing. Owing to its excellent performance, we were able to detect basal levels of Cu in healthy wildtype mice, as well as elevated Cu in a Wilson’s disease model and in a liver metastasis model. To showcase the potential impact of PACu-1 for BFA, we conducted a blind study where we were able to successfully identify a Wilson’s disease animal from a group of healthy control mice with greater than 99.7% confidence.</p>