Background: Contrast-enhanced ultrasonography with perflubutane microbubbles improves the diagnostic accuracy to differentiate benign and malignant focal liver lesions in dogs.Hypothesis: Perflubutane microbubbles-enhanced ultrasonography is useful for differentiation of benign from malignant focal splenic lesions in dogs.Animals: Twenty-nine clinical dogs with single or multiple focal splenic lesions detected by conventional ultrasonography. Methods: Prospective clinical observational study. Perflubutane microbubbles-enhanced ultrasonography was performed in 29 dogs with focal splenic lesions. Qualitative assessment of the enhancement pattern was performed in the early vascular, late vascular, and parenchymal phases.Results: In the early vascular phase, a hypoechoic pattern was significantly associated with malignancy (P 5 .02) with sensitivity of 38% (95% confidence interval [CI], 25-38%) and specificity of 100% (95% CI, 84-100%). In the late vascular phase, a hypoechoic pattern was significantly associated with malignancy (P 5 .001) with sensitivity of 81% (95% CI, 66-90%) and specificity of 85% (95% CI, 65-95%). There was no significant difference between malignant and benign lesions during the parenchymal phase.Conclusions and Clinical Importance: Hypoechoic splenic nodules in the early and late vascular phases with perflubutane microbubbles-enhanced ultrasonography are strongly suggestive of malignancy in dogs.