<p>Focused ultrasound (FUS) combined with micro/nanobubbles enables transient, localized blood brain barrier opening (BBBO) for targeted drug delivery to the brain. Although imaging-guided FUS methods provide accurate targeting, they require specialized infrastructure that increases cost and workflow complexity. Commercial stereotactic FUS platforms such as the RK-50 offer an accessible alternative, but standard procedures rely on a scalp incision to identify bregma for atlas registration, introducing tissue injury and potential confounds for repeat-BBBO studies. Here, we describe and validate a fully non-invasive targeting approach for FUS BBBO in mice using the RK-50 platform. Our novel targeting method achieved accurate, reproducible targeting without surgical exposure. Evans blue extravasation and atlas co-registration demonstrated sub-millimeter targeting accuracy at bregma and confirmed reliable BBBO in the cortex, hippocampus, and cerebellum. Across these anatomically distinct targets, passive cavitation detection showed broadly comparable acoustic emissions profiles. We further compared this non-invasive workflow with the standard incisional approach in a repeat-BBBO paradigm and observed similar acoustic emissions and comparable delivery of both small molecule and large molecule (antibody) reporters. Together, these findings establish a high-fidelity, incision-free targeting strategy that preserves the accessibility of stereotactic FUS while reducing procedural burden and improving suitability for longitudinal preclinical BBBO studies.</p>