An increased research focus on the lymphatic system has necessitated the development of reliable in vivo assessments of lymphatic function in rodent models. Here, we provide three protocols for assays based upon near-infrared fluorescence imaging that were developed in our laboratory that can visualize and quantify different aspects of lymphatic function in mice. The first, a protocol for the tracking of the clearance of an injected intradermal bolus of a pegylated tracer, provides a measurement of lymphatic function in a specific region of the skin. The second assay allows noninvasive imaging of the contractility of collecting lymphatic vessels of the lower limb after injection of the pegylated tracer into the paw skin. This assay also enables real-time visualization of the routing of lymphatic flow from the paw to draining lymph nodes. The final protocol describes invasive imaging of the contractility and valve function of a collecting lymphatic vessel connecting the inguinal and axillary lymph nodes. This assay allows compounds to be added directly on the collecting lymphatic vessel and responses in contraction frequency and amplitude to be measured.