Clearance of brain waste in the cerebrospinal fluid (CSF) through the meningeal lymphatic vessels (mLV) has been evaluated mostly through the fluorescent imaging which has inherent limitations in the context of animal physiology and clinical translatability. The study aimed to establish molecular imaging for the evaluation of mLV clearance function. Radionuclide imaging after intrathecal (IT) injection was acquired in C57BL/6 mice of 2-9 months. The distribution of [99mTc]Tc-diethylenetriamine pentaacetate (DTPA) and [64Cu]Cu-human serum albumin (HSA) was comparatively evaluated. Evans Blue and [64Cu]Cu-HSA were used to evaluate the distribution of tracer under various speed and volume conditions. [99mTc]Tc-DTPA is not a suitable tracer for evaluation of CSF clearance via mLV as no cervical lymph node uptake was observed while it was cleared from the body. A total volume of 3 to 9 μL at an infusion rate of 300 to 500 nL/min was not sufficient for the tracer to reach the cranial subarachnoid space and clear throughout the mLV. As a result, whole-body positron emission tomography imaging using [64Cu]Cu-HSA at 700 nL/min, to deliver 6 μL of injected volume, was set for characterization of the CSF to mLV clearance. Through this protocol, the mean terminal CSF clearance half-life was measured to be 123.6 min (range 117.0-135.0) in normal mice. We established molecular imaging to evaluate CSF drainage through mLV using [64Cu]Cu-HSA. This imaging method is expected to be extended in animal models of dysfunctional meningeal lymphatic clearance and translational research for disease-modifying therapeutic approaches. The online version contains supplementary material available at 10.1007/s13139-022-00746-6.