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OBJECTIVE To develop an innovative process for stereotactic brain biopsies in dogs and cats that would provide a definitive diagnosis and optimize the management of patients with brain lesions. ANIMALS 4 dogs and 1 cat diagnosed with 1 or more brain lesion(s) underwent brain biopsies between March 24, 2023, and October 25, 2023. METHODS Based on trajectories selected on images of MRI and CT scan performed on each patient, a computerized software program was used to design a 3-D–printed patient-specific device with maxillary dental impression located on a baseplate to secure the patient’s head and with insertion ports for the biopsy instrumentations located on a C-arm. As proof of concept, the device was successfully used in 2 cadavers before being used on clinical patients. All biopsy samples were submitted for histopathological examination. RESULTS Histological diagnosis was obtained in 80% (4/5) of the cases (choroid plexus tumor, astrocytoma, meningioma, and chronic meningoencephalitis of unknown origin). In 1 patient, the results of biopsy were nondiagnostic; postmortem diagnosis was consistent with a low-grade oligodendroglioma. All the patients were discharged within 24 hours after the procedure without complications. This novel stereotactic system allows the surgeon to perform safe, easyto-use, inexpensive, and minimally invasive precise brain biopsies in dogs and cats, without complications. CLINICAL RELEVANCE This unique technique could be applied to any size and type of skull and for any type of brain lesions and would provide diagnostic information that would be valuable for future treatment planning and prognosis. [ABSTRACT FROM AUTHOR]