The development of cell-type-specific gene therapy vectors for treating neurological diseases holds great promise, but has relied on animal models with limited translational utility. We have adapted an ex vivo organotypic model to evaluate adeno-associated virus (AAV) transduction properties in living slices of human brain tissue. Using fluorescent reporter expression and single-nucleus RNA sequencing, we found that common AAV vectors show broad transduction of normal cell types, with protein expression most apparent in astrocytes; this work introduces a pipeline for identifying and optimizing AAV gene therapy vectors in human brain samples.