AbstractCombretastatin A4-phosphate (CA4P) is an anti-vascular agent which selectively shuts down blood supply in tumours, resulting in extensive tumour necrosis. The aim of this study was to assess in vivo, non-invasive ultrasound techniques for the early evaluation of tumour perfusion following CA4P treatment of spontaneous tumours. Eight dogs that bore spontaneous tumours were enrolled and were subsequently treated with a single dose of intravenous CA4P. Perfusion of tumours was evaluated by power Doppler ultrasound (PDUS) pre-treatment (0 h), during the injection (10 min, 20 min, 30 min) and after CA4P infusion (24 and 72 h). Vascularity index (VI) of the tumour tissue was quantitatively analysed and accuracy was verified by correlation analysis with the results of immunohistochemical evaluation of microvessel density (MVD). Central and peripheral perfusion was evaluated by contrast-enhanced ultrasound (CEUS) pre-treatment and at 72 h post-treatment. Post-treatment, PDUS demonstrated a significant decrease in VI within 10 min of CA4P infusion. CEUS parameters demonstrated a significant decrease in blood velocity and volume in the central aspect of the tumour. Histology revealed a 4.4-fold reduction (p < 0.001, 95% CI [2.2,9.4]) in MVD and a 4.1-fold increase (p = 0.003, 95% CI [1.4,11.8]) in necrotic tumour tissue. A strong correlation between PDUS results and immunohistochemical results was found (Pearson R² = 0.957, p < 0.001). Furthermore, the findings of PDUS were supported by the objective results of the CEUS analyses. These data suggest a role for ultrasound in real-time, non-invasive monitoring of tumour vascular response as an early indicator of CA4P treatment efficacy.