Purpose: Urokinase-like Plasminogen Activator Receptor (uPAR) is overexpressed in a variety of carcinoma types, and therefore represents an attractive imaging target. The aim of this study was to assess the feasibility of two uPAR-targeted probes for PET and fluorescence tumor imaging in a human xenograft tongue cancer model. Experimental design and results: Tumor growth of tongue cancer was monitored by bioluminescence imaging (BLI) and MRI. Either ICG-Glu-Glu-AE105 (fluorescent agent) or 64Cu-DOTA-AE105 (PET agent) was injected systemically, and fluorescence imaging or PET/CT imaging was performed. Tissue was collected for micro-fluorescence imaging and histology. A clear fluorescent signal was detected in the primary tumor with a mean in vivo tumor-to-background ratio of 2.5. Real-time fluorescence-guided tumor resection was possible, and sub-millimeter tumor deposits could be localized. Histological analysis showed co-localization of the fluorescent signal, uPAR expression and tumor deposits. In addition, the feasibility of uPARguided robotic cancer surgery was demonstrated. Also, uPAR-PET imaging showed a clear and localized signal in the tongue tumors. Conclusions: This study demonstrated the feasibility of combining two uPARtargeted probes in a preclinical head and neck cancer model. The PET modality provided preoperative non-invasive tumor imaging and the optical modality allowed for real-time fluorescence-guided tumor detection and resection. Clinical translation of this platform seems promising.