Microvascular impairments are a major issue in many diseases of the brain. In fact, they are often considered the earliest pathological phenomena in neurodegenerative diseases like Alzheimer's Disease or the locus of pathology as in stroke. Still, little is known about the mechanistic and cellular level events that contribute to these impairments. Given the importance of the neurovascular unit (NVU) in maintaining functional brain tissue, alterations to NVU cell types are important to study in the context of disease progression. With the use of two-photon microscopy, microvessels and cells can be imaged and evaluated throughout disease progression. Herein we aim to provide a comprehensive protocol for setting up and using two-photon microscopy for serial imaging of neurovascular unit cell types (i.e., pericytes, astrocytes, and microglia). We also describe interpreting results from cell and vessel changes based on disease or functional tests through multiple timepoints. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mouse set up for serial, live 2-photon imaging Basic Protocol 2: Field of view acquisitions over multiple timepoints Basic Protocol 3: Line scan acquisitions for acquiring red blood cell metrics Basic Protocol 4: Quantitative measurements of vessel and cell changes Basic Protocol 5: Documenting changes in astrocyte morphology following induction of transient focal photothrombotic stroke using Rose Bengal.