Background: Although most patients with PDAC experience distant failure after resection, a significant portion still present with local recurrence. Intraoperative fluorescent imaging can potentially facilitate the visualization of involved peritumoral LNs and guide the locoregional extent of nodal dissection. Here, the efficacy of targeted intraoperative fluorescent imaging was examined in the detection of metastatic lymph nodes (LNs) during resection of pancreatic ductal adenocarcinoma (PDAC). Methods: A dose-escalation prospective study was performed to assess feasibility of tumor detection within peripancreatic LNs using cetuximab-IRDye800 in PDAC patients. Fluorescent imaging of dissected LNs was analyzed ex vivo macroscopically and microscopically and fluorescence was correlated with histopathology. Results: A total of 144 LNs (72 in the low-dose and 72 in the high-dose cohort) were evaluated. Detection of metastatic LNs by fluorescence was better in the low-dose (50 mg) cohort, where sensitivity and specificity was 100% and 78% macroscopically, and 91% and 66% microscopically. More importantly, this method was able to detect occult foci of tumor (measuring < 5 mm) with a sensitivity of 88% (15/17 LNs). Conclusion: This study provides proof of concept that intraoperative fluorescent imaging with cetuximab-IRDye800 can facilitate the detection of peripancreatic lymph nodes often containing subclinical foci of disease.