Pompe disease (PD) is a rare autosomal-recessive glycogen storage disorder resulting in proximal muscle weakness and loss of respiratory function. While enzyme replacement therapy (ERT) is the only effective treatment, biomarkers for disease monitoring are scarce. After ex vivo biomarker validation in phantom studies, we applied multispectral optoacoustic tomography (MSOT), a molecular sensitive ultrasound approach, in a clinical trial (NCT05083806) to image biceps muscles of 10 late-onset PD patients (LOPD) compared to matched healthy controls. MSOT was compared to muscle magnetic resonance imaging (MRI), ultrasound, spirometry, muscle testing, and quality of life score (QOL). Additionally, the results were validated in an independent LOPD patient cohort from a second clinical site. Our study demonstrated that MSOT enabled imaging of subcellular disease pathology with increases in glycogen/water, collagen and lipid signals providing higher sensitivity to detect muscle degeneration than current clinical and imaging methods. This translation approach suggests implementation in the complex care of these ultra-rare disease patients.