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Summary: The endothelial glycocalyx is an integral component of the brain vascular barrier. Visualizing its structure in vivo is essential to understand its physiological and pathophysiological mechanisms. Here, we present a surgical protocol for chronic cranial window implantation in mice, alongside the use of multiphoton microscopy tools to image the cortical vasculature. We describe steps for cranial window implantation, intravenous injection of fluorescent markers, and intravital imaging. We then detail a technique to quantify glycocalyx thickness using Imaris image analysis software.For complete details on the use and execution of this protocol, please refer to Gray et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.