The goal of our studies was to determine lymphatic uptake of bovine serum albumin (BSA) using real-time noninvasive fluorescence imaging. BSA labeled with near-infrared dye (IRDye) 680 was used as a model protein-dye conjugate. The conjugation of BSA with IRDye 680 was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Size-exclusion high-performance liquid chromatography and SDS-PAGE demonstrated that the IRDye 680-labeled BSA conjugate in the lymph node (LN) homogenate samples was stable at physiological temperature (37°C) for at least 5 days. Whole-body noninvasive optical imaging of hairless SKH-1 mice was performed after subcutaneous (s.c.) injection (dose = 0.1 mg/kg) into the front footpad. Noninvasive fluorescence imaging demonstrated that BSA-IRDye 680 conjugates were dynamically taken up by the lymphatic system, accumulated in the axillary LNs and then cleared, indicating that lymphatic transport plays a role in the absorption of BSA. Ex vivo tissue imaging of LN homogenates provided confirmatory data with respect to the uptake of fluorescent-labeled BSA determined by in vivo imaging. Noninvasive real-time imaging of LNs provides a novel tool for evaluating uptake and accumulation of fluorescent-labeled proteins by the lymphatic system after s.c. injection in a mouse model.