In this study, we applied multiplexed positron emission tomography (PET) probes to monitor glucose metabolism, cellular proliferation, tumor hypoxia and angiogenesis during VEGF₁₂₁/rGel therapy of breast cancer. Two doses of 12 mg/kg VEGF₁₂₁/rGel, administered intraperitoneally, resulted in initial delay of tumor growth, but the growth resumed 4 days after tumor treatment was stopped. The average tumor growth rate expressed as V/V(0), were 1.11 ± 0.07, 1.21 ± 0.10, 1.58 ± 0.36 and 2.64 ± 0.72 at days 1, 3, 7 and 14, respectively. Meanwhile, the VEGF₁₂₁/rGel treatment group showed V/V₀ ratios of 1.04 ± 0.06, 1.05 ± 0.11, 1.09 ± 0.17 and 1.86 ± 0.36 at days 1, 3, 7 and 14, respectively. VEGF₁₂₁/rGel treatment led to significantly decreased uptake of ¹⁸F-FPPRGD2 at day 1 (24.0 ± 8.8%, p < 0.05) and day 3 (36.3 ± 9.2%, p < 0.01), relative to the baseline, which slowly recovered to the baseline at day 14. ¹⁸F-FMISO uptake was increased in the treated tumors at day 1 (23.9 ± 15.7%, p < 0.05) and day 3 (51.4 ± 29.4%, p < 0.01), as compared to the control group. At days 7 and 14, ¹⁸F-FMISO uptake restored to the baseline level. The relative reductions in FLT uptake in treated tumors were approximately 13.0 ± 4.5% at day 1 and 25.0 ± 4.4% (p < 0.01) at day 3. No significant change of ¹⁸F-FDG uptake was observed in VEGF₁₂₁/rGel treated tumors, compared with the control group. The imaging findings were supported by ex vivo analysis of related biomarkers. Overall, longitudinal imaging studies with 4 PET tracers demonstrated the feasibility and usefulness of multiplexed probes for quantitative measurement of antitumor effects of VEGF₁₂₁/rGel at the early stage of treatment. This preclinical study should be helpful in accelerating anticancer drug development and promoting the clinical translation of molecular imaging.