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OBJECTIVE To assess the accuracy of transsphenoidal hypophysectomy using 3-D printed patient-specific guides (3D-PSGs) in small-breed dogs. ANIMALS Heads obtained from the cadavers of 19 small-breed dogs (ex vivo portion of study) and 3 healthy adult (3 to 4 years) purpose-bred Beagles with a median body weight of 9.2 kg. PROCEDURES In the ex vivo study, CT images of the cadavers were collected. The position, width, and length of the pituitary fossa and the pilot hole (insertion angle and place) were measured. Using PSGs, 19 pilot holes were made for the pituitary gland fossa, and CT was performed to assess the position accuracy. In the in vivo study, 3 surgical windows from the pilot holes were made using PSGs. Repeated CT and MRI were performed to evaluate the safeness and effectiveness of PSGs, followed by necropsy. RESULTS In the ex vivo study, the median (interquartile range) difference between the pre- and postoperative insertion angles was 2° (0° to 3.5°) and the median deviation of the pilot hole was 0.46 mm (0 to 1.58 mm). In the in vivo study, the surrounding structures were not damaged, and favorable outcomes were evident in terms of the shape, size, and position of the surgical window. CLINICAL RELEVANCE 3D-PSGs provided a safe and effective surgical window for transsphenoidal hypophysectomy. Our findings emphasized the applicability of PSGs in brain surgery, in terms of accuracy and effectiveness.