Understanding the cellular and circuit basis of early activity-dependent brain development is key to addressing basic and translational neuroscience questions.  To achieve this goal, we need to manipulate the activity of specific neuronal subtypes while assaying activity in vivo – but this is a daunting task in newborn mice.  The major bottleneck has been the technical difficulty of delivering, expressing, and utilizing activity manipulation tools in the fragile neonatal mouse.  Adeno-associated viruses (AAV) are a common gene transfer system in adults but generally take several weeks to achieve high expression; thus, they have not been widely used for in vivo studies of very young animals.  We have refined an AAV-mediated approach for optogenetic and chemogenetic tool delivery at early developmental stages.  By injecting AAV into the mouse brain at postnatal day 0 (P0), our approach allows manipulation of neuronal subtypes in the cortex, thalamus, and hippocampus in un-anesthetized neonatal mice as early as P3.  This protocol describes the general guideline of viral-mediated gene delivery for in vivo circuit manipulation in neonatal mice.