Ultrasound-driven microbubbles (USMB) have synergistic effects with radiotherapy (RT). Sequence and timing of RT and vascular-disrupting agents have a large effect on cytotoxicity. We investigated outcomes based on USMB and RT sequencing in a preclinical prostate cancer model. PC-3 prostate cancer xenografts were treated with ultrasound-driven lipid microspheres and 8 Gy of external-beam RT. Sequence 1 (S1) consisted of USMB treatment preceding RT by 3, 6, 12, and 24 hours; Sequence 2 (S2) delivered USMB after RT at the same times. 5 tumors were treated at baseline (no treatment) and at each time point. Tumor effect was assessed via CD31 staining, TUNEL and H&E staining, and Carbonic Anhydrase 9 (CA9) staining to measure microvessel density, cell death and hypoxia, respectively. For the tumors treated using S1, microvessel density (MVD) was 18.8±4.8 counts per field (cpf) at baseline and 9.8±2.3, 9.1±2.5, 11.0±3.9 and 12.8±3.3 cpf when USMB preceded RT by 3, 6, 12 and 24 hours, respectively. Using S2, MVD was 18.0±1.8 cpf at baseline and 12.2±0.7, 10.7±1.1, 12.1±1.9 and 14.2±1.8 cpf, respectively, when RT preceded USMB by the same times. Each MVD measurement was significantly lower than baseline (p<0.05) except for S2 separated by 24 hours (S2-24). The MVD using S1 was larger than S2 at all time points, but none of these differences were statistically significant. Percent cell death (CD) using S1 was 19.2%±4.4% at baseline and 28.0%±6.4%, 35.6%±10.4%, 30.8%±9.0% and 26.4%±3.4% when USMB preceded RT by 3, 6, 12 and 24 hours, respectively. Using S2, CD was 14.8%±4.4% at baseline and 24.2%±7.0%, 27.8%±5.2%, 24.8%±6.1% and 22.4%±5.2%, respectively, at the same times. Only the S1-6 measurement differed significantly from baseline (p<0.01). There was more cell death with S1 than S2 at all time points but these differences were not statistically significant (p>0.05). By both MVD and CD, maximum effect occurred using S1-6. Among S2 measurements, maximum effect was using S2-6. However, despite the clear trend of increasing effect with longer separation time up to 6 hours followed by a decrease with longer separation, the measurements at 3, 6, 12 and 24 hours were not significantly different from each other using either S1 or S2 (p>0.05). The only hypoxia measurement significantly different from baseline was S1-6 (p<0.01). The lowest S2 measurement was S2-6, but it did not differ significantly from baseline (p>0.05). Less hypoxia was measured using S1 than S2 at all time points but these differences were not statistically significant (p>0.05). A trend was seen toward S1 yielding greater tumor effect than S2 and toward maximum tumor effect with treatment separation of 6 hours. However, our sample was not large enough to establish statistical significance. Testing of a larger sample should confirm this finding after which further testing is warranted to develop clinical protocols for combining USMB and RT.