Aquaporins (AQPs) are water channel proteins found throughout the body. In the brain, AQP4 is the most abundant AQP and is highly expressed in astrocytes. Astrocytes regulate brain water homeostasis by relocalising AQP4 from intracellular vesicular pools to the cell surface, with the highest density of AQP4 found at astrocyte endfeet covering cerebral blood vessels. This subcellular relocalisation of AQP4 has a crucial role in the regulation of AQP4 function that is independent of AQP4 expression. The glymphatic system clears waste products and excess fluid from the brain, and glymphatic dysfunction has been implicated in pathological conditions such as Alzheimer’s disease and cerebral oedema. Due to the enrichment of AQP4 at astrocyte endfeet and the reduction of glymphatic function in AQP4-/- mice, AQP4 is thought to have a pivotal role in glymphatic function. Recently, organotypic brain slice cultures (OBSCs) have been used to model diverse diseases in vitro. Although we cannot model the glymphatic system directly in OBSCs, we can study its cellular and structural elements. However, we do not fully understand the effects of the lack of blood flow and intracranial pressure in OBSCs on AQP4 localisation and function in astrocytes. To investigate this, the distribution and localisation of AQP4 were compared between acute, 3-day and 8-day OBSCs from adult rats cultured at the air-liquid interface using a serum-free media formulation. Immunohistochemistry was performed to visualise and quantify differences in the expression and localisation of AQP4 in astrocytes between acute and cultured OBSCs. We further established that the OBSC model can be used to measure changes in AQP4 localisation in response to treatments that increase the membrane water permeability of cultured primary human astrocytes. Understanding the changes in AQP4 localisation in response to the lack of blood flow in the OBSC model will aid in future research to investigate elements of the glymphatic system and contribute to the development of therapies to promote fluid clearance. Using the OBSC model to measure changes in AQP4 localisation to the astrocyte endfeet following drug treatments could be a useful method to identify potential glymphatic-enhancing drugs for further study in vivo.