To evaluate if clinical fluorescence imaging of IRDye800CW is feasible on our fluorescence optical mammography system by estimating detection limits assessed by breast-cancer-simulating phantom experiments. Phantoms (2.1 cm(3), 0.9 cm(3)) with IRDye800CW concentrations of 0.5 to 120 nM were suspended in a 550 cm(3) measurement cup containing 507 surface-mounted source and detector fibers. The cup was filled with optical matching fluid containing IRDye800CW concentrations of 0, 5, 10, or 20 nM. Tomographic fluorescence images were acquired by exciting IRDye800CW at 730 nm; wavelengths above 750 nm were filtered. Signal intensities were calculated over a volume of interest corresponding to the size and location of the phantom in the reconstructed images. Correlations (R(2)) were calculated, and detection limits with associated upper 95% prediction interval were estimated. Between-day reproducibility was assessed with intraclass correlation coefficients (ICC). Fluorescent intensities were strongly correlated with phantom IRDye800CW concentrations (R(2)0.983 to 0.999). IRDye800CW detection limits ranged from 0.14 to 2.46 nM (upper 95% prediction limit 4.63 to 18.63 nM). ICC ranged from 0.88 to 1.00. The estimated detection limits for IRDye800CW were in the low-nanomolar range. These results support the start of clinical trials to evaluate the fluorescence optical mammography system using IRDye800CW labeled breast cancer targeting ligands.