Morbidity and complexity involved in lymph node staging via surgical resection and biopsy could ideally be improved using node assay techniques that are non-invasive. While visible blue dyes are often used to locate the sentinel lymph nodes from draining lymphatic vessels near a tumor, they do not provide an in situ metric to evaluate presence of cancer. In this study, the transport kinetics of methylene blue were analyzed to determine the potential for better in situ information about metastatic involvement in the nodes. A rat model with cancer cells in the axillary lymph nodes was used, with methylene blue injection to image the fluorescence kinetics. The lymphatic flow from injection sites to nodes was imaged and the relative kinetics from feeding lymphatic ducts relative to lymph nodes was quantified. Large variability existed in raw fluorescence and transport patterns within each cohort resulting in no systematic difference between average nodal uptake in normal, sham control and cancer-bearing nodes. However, when the signal from the afferent lymph vessel fluorescence was used to normalize the signal of the lymph nodes, the high signal heterogeneity was reduced. Using a model, the lymph flow through the nodes (FLN) was estimated to be 1.49 ± 0.64 ml/g/min in normal nodes, 1.53 ± 0.45 ml/g/min in sham control nodes, and reduced to 0.50 ± 0.24 ml/g/min in cancer-cell injected nodes. This summarizes the significant difference (p = 0.0002) between cancer-free and cancer-bearing nodes in normalized flow. This process of normalized flow imaging could be used as an in situ tool to detect metastatic involvement in nodes.