© 2016, World Molecular Imaging Society. Purpose: Intraoperative optical imaging to guide surgeons during oncologic resections offers a unique and promising solution to the ambiguity of cancer margins to tactile and visual assessment that results in devastatingly high rates of positive margins. Sequestering of labeled antibodies by normal tissues with high expression of the antibody target, or “antigen sinks”, diminishes the efficacy of these probes to provide contrast between the tumor and background tissues by decreasing the amount of circulating probe available for uptake by the tumor and by increasing the fluorescence of non-tumor tissues. We hypothesized that administering a dose of unlabeled antibody prior to infusion of the near-infrared (NIR) fluorescently labeled antibody would improve tumor-specific uptake and contrast of the fluorescently labeled probe by occupying extra-tumoral binding sites, thereby increasing the amount of labeled probe available for uptake by the tumor. Procedures: In this study, we explore this concept by testing two different “pre-load” doses of unlabeled cetuximab (the standard 10-mg test dose, and a larger, experimental 100-mg test dose) in six patients receiving cetuximab conjugated to the fluorescent dye IRDye800CW (cetuximab-IRDye800CW) in a clinical trial, and compared the amount of fluorescent antibody in tumor and background tissues, as well as the tumor-specific contrast of each. Results: The patients receiving the larger preload (100 mg) of unlabeled cetuximab demonstrated significantly higher concentrations (9.5 vs. 0.1 μg) and a longer half-life (30.3 vs. 20.6 days) of the labeled cetuximab in plasma, as well as significantly greater tumor fluorescence (32.3 vs. 9.3 relative fluorescence units) and tumor to background ratios (TBRs) (5.5 vs. 1.7). Conclusions: Administering a preload of unlabeled antibody prior to infusion of the fluorescently labeled drug may be a simple and effective way to improve the performance of antibody-based probes to guide surgical resection of solid malignancies.