The brain’s transport system for cerebrospinal and interstitial fluid, the glymphatic system, was first described in 2012 by the Nedergaard team, operates primarily during sleep, and has been linked to pathological neurological conditions including Alzheimer’s disease, traumatic brain injury (TBI), and stroke. Obtaining quantitative measurements of glymphatic fluid velocity and pressure is crucial to understanding the function, failures, and potential rehabilitation of the glymphatic system. However, existing techniques for obtaining in vivo glymphatic velocities are limited to sparse measurements and specific regions, and pressure variation is essentially impossible to measure in vivo. We propose to quantify glymphatic flows from measurements of tracked particles and contrast agents using physics-informed neural networks (PINNs), which can infer velocity and pressure from sparse measurements and have not been used previously in neuroscience. We will adapt PINNs for three commonly-employed glymphatic imaging modalities: two-photon perivascular space imaging, transcranial whole-brain imaging, and dynamic contrast-enhanced magnetic resonance imaging (DCEMRI). For these modalities, each of which can probe different regions and scales of glymphatic flows, we will adapt the PINNs equations and artificial intelligence hyperparameters, evaluate the sensitivity of the approach to noise, spatiotemporal resolution, and imaging artifacts using synthetic data, and validate by comparing velocities inferred by PINNs to velocities from alternative techniques. Using PINNs will allow us to obtain in vivo velocity and pressure measurements of cerebrospinal fluid in previously unmeasured regions of the brain. Our collaborative team of neuroscientists, fluid dynamicists, and applied mathematicians includes the leaders who discovered the glymphatic system and invented PINNs. Moreover, we have extensive experience with all three imaging modalities and with velocity measurement (via automated particle tracking and front tracking) in glymphatic flows. This proposal seeks to reveal mechanisms by which the brain's transport system for cerebrospinal and interstitial fluid operates. Our novel velocity and pressure measurements of intracranial cerebral spinal fluid flows may demonstrate how improving sleep, the state during which the glymphatic system primarily operates, can counteract pathological processes related to glymphatic system failure including Alzheimer's disease.