Introduction Retention of 2-deoxy-2-[F-18]fluoro-D-glucose F-18-FDG in the bladder causes more problems in small animal research than in human research owing to the smaller size of the subject. Catheterization has been proposed to reduce bladder spillover both in human studies and in small animal research. Noninvasive alternatives such as hydration plus furosemide also seem to be a promising pre-imaging strategy for decreasing bladder spillover. Our main goal was to measure the effects of the combination of furosemide and hydration for reducing bladder signal directly on mouse bowel F-18-FDG-PET images. Methods Nine mice were divided into two groups: the control group (C, n = 4) and the treatment group (n = 5). The clearance protocol combines hyperhydration and a single furosemide dose during the F-18-FDG uptake period. Two images were acquired on different days in treated mice to evaluate two different furosemide doses (low dose, LD, 3.5 mg/kg; and high dose, HD, 7 mg/kg). A region of interest was drawn on each computed tomography image (bladder, kidneys, liver, muscle, and bone marrow). To quantify bladder spillover, two different areas of the colon were selected. Results A remarkable reduction in bladder spillover was achieved on F-18-FDG-PET in both groups. Our imaging findings were quantified, and both furosemide doses induced a decrease in mean standard uptake values (SUVmean) compared with the controls (LD 1.46 +/- 0.54 and HD 1.05 +/- 0.29; controls: 8.90 +/- 3.4 [p-value < 0.05]). Conclusion We validated a non-invasive, easy, and harmless pre-imaging alternative for decreasing F-18-FDG bladder spillover. Our study shows the effect of furosemide on bladder spillover directly on F-18-FDG-PET images by measuring SUVmean in the bladder, colon, liver, muscle, and bone marrow.