Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific genes. Multiple means of assessing non-coding DNA and various chromatin states have proven useful in predicting the presence of enhancer sequences in the genome, but validating the activity of these sequences and finding the organs and developmental stages they are active in is a labor-intensive process. Recent advances in adeno-associated virus (AAV) vectors have enabled the widespread delivery of transgenes to mouse tissues, enabling in vivo enhancer testing without necessitating a transgenic animal. This protocol shows how a reporter construct that expresses EGFP under the control of a minimal promoter, which does not drive significant expression on its own, can be used to study the activity patterns of candidate enhancer sequences in the mouse brain. An AAV-packaged reporter construct is delivered to the mouse brain and incubated for 1-4 weeks, after which the animal is sacrificed, and brain sections are observed under a microscope. EGFP appears in cells in which the tested enhancer is sufficient to initiate gene expression, pinpointing the location and developmental stage in which the enhancer is active in the brain. Standard cloning methods, low-cost AAV packaging, and expanding AAV serotypes and methods for in vivo delivery and standard imaging readout make this an accessible approach for the study of how gene expression is regulated in the brain.