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Abstract Incomplete surgical resection in head and neck cancer can lead to locoregional recurrence in >35% of patients. Approaches such as image‐guided surgery (IGS) and post‐operative photodynamic therapy (PDT) have been proposed to reduce recurrence rates. However, the PDT doses needed to eliminate all unresected diseases are not established. This in vitro proof‐of‐concept study aims to predict head and neck tumor nodule viability in vitro following PDT with TLD1433 using the IGS probe ABY‐029. ABY‐029 is an EGFR‐specific affibody‐IRDye800CW conjugate that has undergone Phase 0 evaluation studies in head and neck cancer, among others. TLD1433 is a ruthenium‐based photosensitizer in a Phase II trial for non‐muscle invasive bladder cancer. Here, we demonstrate that decreases in fluorescence emission of ABY‐029 bound to MOC1 mouse head and neck cancer nodules in vitro can be predictive of TLD1433 PDT responses. Results show that photoactivation of TLD1433 produces reactive oxygen species (ROS) that reduce MOC1 nodule fractional viability in a manner that is inversely correlated with ABY‐029 fluorescence intensity (Pearson'sr = −0.9148,R² = 0.8369,p < 0.0001). We hypothesize that this is due to ROS‐mediated degradation of IRDye800CW. The findings warrant further studies using head and neck cancer nodules with heterogenous PDT responses and EGFR expression levels. If successful, the future goal would be to use ABY‐029 to guide the dosimetry of intraoperative PDT of the surgical bed after IGS to eliminate all microscopic diseases, reduce recurrence rates, and prolong survival.