Abstract Purpose: Optimal head and neck squamous cell carcinoma (HNSCC) patient selection for anti–EGFR-based therapy remains an unmet need since only a minority of patients derive long-term benefit from cetuximab treatment. We assessed the ability of state-of-the-art noninvasive in vivo metabolic imaging to probe metabolic shift in cetuximab-sensitive and -resistant HNSCC patient-derived tumor xenografts (PDTXs). Experimental Design: Three models selected based on their known sensitivity to cetuximab in patients (cetuximab-sensitive or acquired-resistant HNC007 PDTXs, cetuximab-naïve UCLHN4 PDTXs, and cetuximab-resistant HNC010 PDTXs) were inoculated in athymic nude mice. Results: Cetuximab induced tumor size stabilization in mice for 4 weeks in cetuximab-sensitive and -naïve models treated with weekly injections (30 mg/kg) of cetuximab. Hyperpolarized 13C-pyruvate–13C-lactate exchange was significantly decreased in vivo in cetuximab-sensitive xenograft models 8 days after treatment initiation, whereas it was not modified in cetuximab-resistant xenografts. Ex vivo analysis of sensitive tumors resected at day 8 after treatment highlighted specific metabolic changes, likely to participate in the decrease in the lactate to pyruvate ratio in vivo. Diffusion MRI showed a decrease in tumor cellularity in the HNC007-sensitive tumors, but failed to show sensitivity to cetuximab in the UCLHN4 model. Conclusions: This study constitutes the first in vivo demonstration of cetuximab-induced metabolic changes in cetuximab-sensitive HNSCC PDTXs that were not present in resistant tumors. Using metabolic imaging, we were able to identify hyperpolarized 13C-pyruvate as a potential marker for response and resistance to the EGFR inhibitor in HNSCC.