© 2017 Wiley Periodicals, Inc. Background and Objectives: Optical imaging to guide cancer resections is rapidly transitioning into the operating room. However, the sensitivity of this technique to detect subclinical disease is yet characterized. The purpose of this study was to determine the minimum range of cancer cells that can be detected by antibody-based fluorescence imaging. Methods: 2LMP (breast), COLO-205 (colon), MiaPaca-2 (pancreas), and SCC-1 (head and neck) cells incubated in vitro with cetuximab-IRDye800CW (dose range 8.6-86 nM) were implanted subcutaneously in mice (n = 3 mice, 5 tumors/mouse). Following incubation with 8.6 × 10−2 µM of cetuximab-IRDye800CW in vitro, serial dilutions of each cell type (1 × 103–1 × 106) were implanted subcutaneously (n = 3, 5 tumors/mouse). Tumors were imaged with Pearl Impulse and Xenogen IVIS 100 imaging systems. Scatchard analysis was performed to determine receptor density and kinetics for each cell line. Results: Under conditions of minimal cetuximab-IRDye800CW exposure to low cellular quantity, closed-field fluorescence imaging theoretically detected a minimum of 4.2 × 104-9.5 × 104 2LMP cells, 1.9 × 105-4.5 × 105 MiaPaca-2 cells, and 2.4 × 104-6.7 × 104 SCC-1 cells; COLO-205 cells could not be identified. Higher EGFR-mediated uptake of cetuximab correlated with sensitivity of detection. Conclusion: This study supports the clinical utility of cetuximab-IRDye800CW to sensitively localize subclinical disease in the surgical setting.