Background: Despite the improvement in clinical outcomes for head and neck squamous cell carcinoma (HNSCC) as the result of cetuximab, patients may present with or develop resistance that increases tumor recurrence rates and limits clinical efficacy. Therefore, identifying those patients who are or become resistant is essential to tailor the best therapeutic approach. Materials and Methods: Cetuximab was conjugated to p-NCS-Bz-DFO and labeled with 89Zr. The resistance model was developed by treating FaDu cells with cetuximab. Western blotting (WB) and specific binding assays were performed to evaluate epidermal growth factor receptor (EGFR) expression and 89Zr-DFO-cetuximab uptake in FaDu cetuximab-resistant (FCR) and FaDu cetuximab-sensitive (FCS) cells. Positron emission tomography imaging and biodistribution were conducted in NU/NU nude mice implanted with FCR or FCS cells. Results: Cetuximab was successfully radiolabeled with 89Zr (≥95%). Binding assays performed in FCR and FCS cells showed significantly lower 89Zr-DFO-cetuximab uptake in FCR (p < 0.0001). WB suggests that the resistance mechanism is associated with EGFR downregulation (p = 0.038). This result is in agreement with the low uptake of 89Zr-DFO-cetuximab in FCR cells. Tumor uptake of 89Zr-DFO-cetuximab in FCR was significantly lower than FCS tumors (p = 0.0340). Conclusions: In this work, the authors showed that 89Zr-DFO-cetuximab is suitable for identification of EGFR downregulation in vitro and in vivo. This radiopharmaceutical may be useful for monitoring resistance in HNSCC patients during cetuximab therapy.